Heteroaryl amides as inhibitors of protein aggregation

ABSTRACT

The present invention relates to certain heteroaryl amide compounds, pharmaceutical compositions containing them, and methods of using them, including methods for preventing, reversing, slowing, or inhibiting protein aggregation, and methods of treating diseases that are associated with protein aggregation, including neurodegenerative diseases such as Parkinson&#39;s disease, Alzheimer&#39;s disease, Lewy body disease, Parkinson&#39;s disease with dementia, fronto-temporal dementia, Huntington&#39;s Disease, amyotrophic lateral sclerosis, and multiple system atrophy, and cancer and melanoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2015/013263, filed Jan. 28, 2015, which claims the benefit of U.S.Provisional Applications No. 61/933,246, filed Jan. 29, 2014, and No.62/078,895, filed Nov. 12, 2014, the contents of which are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The present invention relates to certain heteroaryl amide derivatives,pharmaceutical compositions containing them, and methods of using them,including methods for preventing, reversing, slowing, or inhibitingprotein aggregation, and methods of treating diseases that areassociated with protein aggregation, including neurodegenerativediseases such as Parkinson's disease, Alzheimer's disease, Lewy bodydisease, Parkinson's disease with dementia, fronto-temporal dementia,Huntington's Disease, amyotrophic lateral sclerosis, and multiple systematrophy, and cancer.

SEQUENCE LISTING

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 699462000801SeqList.TXT;date recorded: Dec. 29, 2015; size: 1 KB).

BACKGROUND

Neurodegenerative disorders of the aging population such as Alzheimer'sdisease (AD), Parkinson's disease (PD), and fronto-temporal dementia(FTD), affect over 20 million people in the United States and EuropeanUnion alone and rank among the top causes of death for the elderly. Acommon feature among these neurological disorders is the chronicaccumulation of proteins into neurotoxic aggregates. Each disease ischaracterized by the specific neuronal populations that are affected,the particular protein aggregates that are involved, and the clinicalfeatures that result from the neuronal degeneration.

Studies suggest that the initial stages of protein aggregation involvemutation or post-translational modification (e.g., nitrosilation,oxidation) of the target protein, which then adopts an abnormalconformation that facilitates interactions with similarly misfoldedproteins. The abnormal proteins then aggregate to form dimers, trimers,and higher-order multimers, also termed “soluble oligomers,” which maydisrupt synaptic function. Additionally, the aggregates may then anchorin the cell membrane and form globular oligomers (which in turn can formpores in the membrane) and/or protofibrils or fibrils. These larger,insoluble fibrils may function as reservoirs of the bioactive oligomers.

Diverse lines of evidence support the notion that the progressiveaccumulation of protein aggregates is causally involved in thepathogenesis of neurodegenerative diseases. A number of other proteinsmay accumulate in the brains of patients with neurodegeneration, such asalpha-synuclein, Aβ protein, Tau, and TDP43. The cognitive impairment ofthese patients is closely associated with synaptic loss in the neocortexand limbic systems and increasing levels protein aggregates maycontribute to this synaptic loss. Much research is focused on detailingthe mechanisms through which accumulation of alpha-synuclein and otheramyloid precursor proteins (APP) metabolites contributes to synapticdamage and neurodegeneration. Many studies support the hypothesis thatformation of small aggregates, also known as oligomers, plays a majorrole in neurotoxicity. These peptide oligomers can organize into dimers,trimers, tetramers, pentamers, and other higher order arrays that canform annular structures. High levels of such oligomers are predictive ofdementia and synaptic loss in patients. Because evidence indicates theoligomers rather than smaller precursor fibrils are the toxic species,compounds that target these early aggregation processes in a specificmanner would be useful as potential new therapies for PD, AD and relatedconditions.

Various neurodegenerative diseases involve the accumulation ofneurotoxic protein-based aggregates. In idiopathic Parkinson's disease(IPD), dementia with Lewy bodies (LBD), Parkinson's disease withdementia (PDD), and multiple system atrophy (MSA), the neurotoxicaggregates are composed of α-synuclein (SYN), which is a synapticprotein that is intracellular under normal conditions. In FTD andamyotrophic lateral sclerosis (ALS), neurotoxic aggregates originatefrom other intracellular proteins such as tau, TDP-43, or SOD1. Forcertain diseases, such as AD, SYN aggregates with the primary protein(e.g., Aβ protein). In Huntington's Disease, aggregates form from thecleavage products of Htt proteins.

Accumulation of α-synuclein has also been implicated in cancer, inparticular, in melanoma cancer cells. Pan et al., PLoS One 2012, 7(9),e45183. Thus, compounds that inhibit such accumulation may prove usefulin treatment of various cancers, including melanoma.

Two mechanisms are implicated in these protein aggregation processes. Inthe first, the misfolded and/or aggregated proteins anchor to thevarious cell membrane structures. Binding of the misfolded or aggregatedmolecules to the plasma membrane or the membranes of organelles (e.g.,mitochondria or lysosomes) may interfere with protein transcription,autophagy, mitochondrial function, and pore formation. By way ofexample, neurotoxic SYN aggregates and interacts with lipids in cellmembranes by a specific portion of the c-terminal region of thesynuclein protein. Compounds that bind to this region can inhibitprotein-protein or protein-lipid interactions and can therefore be usedto block neurotoxic oligomerization of SYN or other proteins and theirinteractions with membranes. In the second process, aggregated proteinis released from the anchored subunit and propagates to adjacent cells.This cell-to-cell propagation of toxic protein aggregates may thenunderlie the anatomic progression of neurodegeneration and worsening ofsymptoms. Small molecule drugs that interact with the target proteinsmay limit release and/or propagation, and therefore reduce theneurotoxic effects of aggregated proteins.

Compounds that are inhibitors of protein aggregation are described inPCT Publ. Nos. WO2011/084642, WO2013/148365, WO2013/134371, and PCTAppln. No. PCT/US2013/050719.

There remains a need for inhibitors of protein aggregation withdesirable pharmaceutical properties. Certain heteroaryl amide compoundshave been found in the context of this invention to have proteinaggregation modulating activity.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a chemical entity of thefollowing Formula (I):

wherein

-   R¹ is H, halo, C₁₋₄alkyl, or CF₃;-   R² is H, —CF₃, or C₁₋₄alkyl unsubstituted or substituted with halo    or —CF₃;-   A is a 5-membered heteroaryl ring;-   Y is absent or is C₁₋₄alkylene;-   R³ and R⁴ taken together with the nitrogen to which they are    attached form a monocyclic heterocycloalkyl ring, unsubstituted or    substituted with C₁₋₄alkyl; or where Y is C₁₋₄alkylene, R³ and Y    taken together with the nitrogen to which R³ is attached form a    monocyclic heterocycloalkyl ring, and R⁴ is H or C₁₋₄alkyl;-   or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of Formula (I) is a compoundselected from those species described or exemplified in the detaileddescription below.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising at least one compound of Formula (I) or apharmaceutically acceptable salt thereof. Pharmaceutical compositionsaccording to the invention may further comprise a pharmaceuticallyacceptable excipient. The invention is also a compound of Formula (I) ora pharmaceutically acceptable salt thereof for use as a medicament.

In another aspect, the invention is directed to a method of treating aneurodegenerative disease or condition associated with protein orpeptide aggregation comprising administering to a subject in need ofsuch treatment an effective amount of at least one compound of Formula(I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention is directed to a method of treating adisease or medical condition associated with protein or peptideaggregation, comprising administering to a subject in need of suchtreatment an effective amount of at least one compound of Formula (I) ora pharmaceutically acceptable salt thereof. The invention is alsodirected at use of a compound of Formula (I) in the preparation of amedicament for the treatment of such diseases and medical conditions,and the use of such compounds and salts for treatment of such diseasesand medical conditions.

In yet another aspect, the invention relates to a method of interferingwith the accumulation of protein or peptide aggregates in a cell, orpreventing, slowing, reversing, or inhibiting protein or peptideaggregation in a cell, comprising contacting the cell with an effectiveamount of at least one compound of Formula (I) or a salt thereof, and/orwith at least one pharmaceutical composition of the invention, whereinthe contacting is in vitro, ex vivo, or in vivo.

Additional embodiments, features, and advantages of the invention willbe apparent from the following detailed description and through practiceof the invention.

For the sake of brevity, the disclosures of the publications cited inthis specification, including patents, are herein incorporated byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of Biological Example 3. In FIG. 1A, the Y axis(I/Io) is the ratio of the heteronuclear single quantum coherence (HSQC)spectroscopy signal intensity for ASYN (average of residues 3-23) in thepresence (I) or absence (Io) of lipid membranes. In FIG. 1B, the averageI/Io ratio of ASYN residues 3-23 was plotted as a function of theconcentration of Example 1 added.

FIG. 2 shows the quantification of electron microscopic images of ASYNoligomers in the absence and presence of Example 1, as described inBiological Example 4.

FIG. 3 shows the effect of Example 1 on the accumulation of ASYN in B103neuroblastoma cells expressing GFP-tagged human ASYN, as described inBiological Example 5.

FIG. 4 shows the results of Biological Example 6A and the effects ofExample 1 at 1 mg/kg and 5 mg/kg dosing on transgenic mice in the RoundBeam Task model.

FIG. 5 shows the results of the Biological Example 6A dot blot analysisof cerebral and hippocampal brain homogenates using A11 antibody.

FIG. 6 shows the results of Biological Example 6B and the effects ofExample 1 on ASYN immunolabeling in cortical neuropil and neuronal cellbodies in Line 61 ASYN transgenic mice. FIG. 6A reflects the ASYNneuropil arm, and FIG. 6B reflects the neuronal cell body arm of thestudy.

FIG. 7 shows the results of Biological Example 6B and the effects ofExample 1 on immunolabeling of neurodegeneration-related markersincluding tyrosine hydroxylase (FIG. 7A), NeuN (FIG. 7B), and glialfibrillary acidic protein (GFAP; FIG. 7C).

FIG. 8 shows the results of Biological Example 6B and the effects ofExample 1 on sensorimotor impairment in Line 61 ASYN transgenic miceusing the Round Beam Motor Performance assay.

FIG. 9 shows the results of Biological Example 7A and the effects ofExample 1 on fecal boli counts in Line 61 ASYN transgenic mice.

FIG. 10 shows the results of Biological Example 7B and the effects ofExample 1 on cardiac levels of ASYN in Line 61 ASYN transgenic mice.

FIG. 11 shows the results of Biological Example 7C and the effects ofExample 1 on percentage of image areas with ASYN-GFP in the retinae ofPDNG78 transgenic mice.

FIG. 12 shows the results of Biological Example 7C and the effects ofExample 1 on perivascular and nerve terminal GFP labeling in PDNG78transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the terms “including,” “containing,” and “comprising”are used in their open, non-limiting sense.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value. Whenever a yield isgiven as a percentage, such yield refers to a mass of the entity forwhich the yield is given with respect to the maximum amount of the sameentity that could be obtained under the particular stoichiometricconditions. Concentrations that are given as percentages refer to massratios, unless indicated differently.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. This nomenclature has generally beenderived using the commercially-available AutoNom software (MDL, SanLeandro, Calif.), Version 12.0.2.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

Representative Embodiments

In some embodiments of Formula (I), R¹ is H, fluoro, chloro, bromo,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, ortert-butyl. In other embodiments, R¹ is H or fluoro. In otherembodiments, R¹ is H. In other embodiments, R¹ is fluoro.

In some embodiments, R² is H, —CF₃, or is methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl, each unsubstitutedor substituted with fluoro, chloro, bromo, or —CF₃. In otherembodiments, R² is H. In other embodiments, R² is —CF₃ or is C₁₋₄alkyloptionally substituted with halo or —CF₃. In other embodiments, R² isC₃₋₄alkyl, unsubstituted or substituted with fluoro or —CF₃. In otherembodiments, R² is butyl. In other embodiments, R² is propyl substitutedwith —CF₃.

In some embodiments, R² is in the “R” stereochemical configuration. Inother embodiments, R² is in the “S” stereochemical configuration. Inother embodiments, compounds of Formula (I) are stereochemical mixturesat the R² position. In still other embodiments, R² is substantially “R”or substantially “S” stereochemical configuration.

In some embodiments, A is a 5-membered heteroaryl ring with two or threeheteroatom ring atoms. In other embodiments, A is a 5-memberedheteroaryl ring with two non-adjacent heteroatom ring atoms. In stillother embodiments, A is thiazole, thiadiazole, oxazole, imidazole, ortriazole. In still other embodiments, A is thiazole or thiadiazole. Instill other embodiments, A is thiazole.

In some embodiments, Y is absent. In other embodiments, Y is —CH₂—,—CH₂CH₂—, —CH(CH₃)—, —(CH₂)₃—, —C(CH₃)₂—, —(CH₂)₄—, —CH((CH₂)₂CH₃)—,—CH(CH(CH₃)₂)—, —CH(CH₂CH₃)CH₂—, —CH(CH₃)CH(CH₃)—, —CH(CH₃)(CH₂)₂—, or—CH₂CH(CH₃)CH₂—. In other embodiments, Y is —CH₂—, —CH₂CH₂—, or—CH(CH₃)—. In still other embodiments, Y is —CH₂CH₂—.

In some embodiments, R³ and R⁴ are taken together with the nitrogen towhich they are attached form a monocyclic heterocycloalkyl ring,unsubstituted or substituted with C₁₋₄alkyl. In other embodiments, R³and R⁴ taken together with the nitrogen to which they are attached formazetidine, pyrrolidine, piperidine, azepine, piperazine, morpholine,thiomorpholine, or 1,1-dioxo-thiomorpholine, each unsubstituted orsubstituted with C₁₋₄alkyl. In other embodiments, R³ and R⁴ takentogether with the nitrogen to which they are attached form piperazine,morpholine, or pyrrolidine, each unsubstituted or substituted withC₁₋₄alkyl. In other embodiments, R³ and R⁴ taken together with thenitrogen to which they are attached form piperazine or morpholine, eachunsubstituted or substituted with C₁₋₄alkyl. In other embodiments, R³and R⁴ taken together with the nitrogen to which they are attached formpiperazine, unsubstituted or substituted with C₁₋₄alkyl. In still otherembodiments, R³ and R⁴ taken together with the nitrogen to which theyare attached form piperazine or 4-methyl-piperazine.

In other embodiments, where Y is C₁₋₄alkylene, R³ and Y are takentogether with the nitrogen to which R³ is attached form a monocyclicheterocycloalkyl ring, and R⁴ is H or C₁₋₄alkyl. In other embodiments, Yand R³ taken together with the nitrogen to which R³ is attached formpyrrolidine or piperidine. In other embodiments, R⁴ is H or methyl.

In some embodiments, R¹ is H, R² is H or C₁₋₄alkyl (or is H, or isC₃₋₄alkyl), A is thiazole, Y is absent or is ethylene (or is absent, oris ethylene), and R³ and R⁴ taken together with the nitrogen to whichthey are attached form N-methylpiperazine.

In other embodiments, the compound of Formula (I) is selected from thegroup consisting of:

Ex. Structure Chemical Name  1

N-(1-(1H-Indol-3-yl)hexan-2- yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide  2

N-(2-(1H-Indol-3-yl)ethyl)-2-(4- methylpiperazin-1-yl)thiazole-5-carboxamide  3

N-(1-(1H-Indol-3-yl)hexan-2- yl)-2-(piperazin-1-yl)thiazole-5-carboxamide  4

N-(2-(1H-Indol-3-yl)ethyl)-2-(2- (4-methylpiperazin-1-yl)ethyl)thiazole-5-carboxamide  5

N-(1-(1H-Indol-3-yl)hexan-2- yl)-2-(2-(4-methylpiperazin-1-yl)ethyl)thiazole-5-carboxamide  6

N-(1-(1H-Indol-3-yl)hexan-2- yl)-2-(4-methylpiperazin-1-yl)oxazole-5-carboxamide  7

N-(1-(1H-Indol-3-yl)hexan-2- yl)-5-(4-methylpiperazin-1-yl)-1,3,4-thiadiazole-2-carboxamide  8

N-(1-(1H-Indol-3-yl)hexan-2- yl)-5-(4-methylpiperazin-1-yl)-4H-1,2,4-triazole-3-carboxamide  9

N-(1-(1H-Indol-3-yl)hexan-2- yl)-2-(4-methylpiperazin-1-yl)-1H-imidazole-5-carboxamide 10

N-(1-(5-Fluoro-1H-indol-3- yl)hexan-2-yl)-2-(2-morpholinoethyl)thiazole-5- carboxamide 11

N-(1-(1H-Indol-3-yl)hexan-2- yl)-2-morpholinothiazole-5- carboxamide 12

N-(1-(1H-indol-3-yl)hexan-2- yl)-2-(pyrrolidin-1-yl)thiazole-5-carboxamide 13

N-(1-(1H-indol-3-yl)hexan-2- yl)-2-(2- morpholinoethyl)thiazole-5-carboxamide 14

N-(1-(5-fluoro-1H-indol-3- yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5- carboxamide 15

N-(1-(1H-Indol-3-yl)hexan-2- yl)-N-methyl-2-(4-methylpiperazin-1-yl)thiazole-5- carboxamide 16

N-(1-(6-fluoro-1H-indol-3- yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5- carboxamide 17

N-(1-(5,6-difluoro-1H-indol-3- yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5- carboxamide 18

2-(4-methylpiperazin-1-yl)-N- (6,6,6-trifluoro-1-(1H-indol-3-yl)hexan-2-yl)thiazole-5- carboxamide 19

N-(1-(1H-indol-3-yl)hexan-2- yl)-5-morpholino-1,3,4-thiadiazole-2-carboxamide 20

N-(1-(1H-indol-3-yl)hexan-2- yl)-5-(2-(4-methylpiperazin-1-yl)ethyl)-1,3,4-thiadiazole-2- carboxamide 21

N-(1-(5-fluoro-1H-indol-3- yl)hexan-2-yl)-5-(2-(4-methylpiperazin-1-yl)ethyl)- 1,3,4-thiadiazole-2-carboxamide 22

N-(1-(1H-indol-3-yl)hexan-2- yl)-2-(1-methylpiperidin-4-yl)thiazole-5-carboxamide 23

N-(1-(1H-indol-3-yl)hexan-2- yl)-5-(pyrrolidin-1-yl)-1,3,4-thiadiazole-2-carboxamide 24

N-(1-(1H-indol-3-yl)hexan-2- yl)-N-methyl-5-(4-methylpiperazin-1-yl)-1,3,4- thiadiazole-2-carboxamide 25

N-(1-(1H-indol-3-yl)hexan-2- yl)-5-(1-methylpiperidin-4-yl)-1,3,4-thiadiazole-2-carboxamide 26

N-(1-(1H-indol-3-yl)hexan-2- yl)-2-(4-methyl-1,4-diazepan-1-yl)thiazole-5-carboxamideand pharmaceutically acceptable salts thereof.

In other embodiments, the compound of Formula (I) is selected from thegroup consisting of:

Ex. Structure Chemical Name 27

(S)-N-(1-(1H-Indol-3-yl)hexan- 2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide 28

(R)-N-(1-(1H-Indol-3-yl)hexan- 2-yl)-2-(4-methylpiperazin-1-yl)thiazol-5-carboxamideand pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of Examples 1-11, and pharmaceutically acceptable saltsthereof. In other embodiments, the compound is a hydrochloride saltform. In other embodiments, the compound is Example 27 or 28, or apharmaceutically acceptable salt thereof. In other embodiments, R² ofFormula (I) is in the (S) stereochemical configuration. In otherembodiments, R² of Formula (I) is in the (R) stereochemicalconfiguration.

Chemical Definitions

The term “alkyl” refers to a straight- or branched-chain alkyl grouphaving from 1 to 12 carbon atoms in the chain. Examples of alkyl groupsinclude methyl (Me), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl,isohexyl, and groups that in light of the ordinary skill in the art andthe teachings provided herein would be considered equivalent to any oneof the foregoing examples.

The term “heteroaryl” refers to a monocyclic, fused bicyclic, or fusedpolycyclic aromatic heterocycle (ring structure having ring atomsselected from carbon atoms and up to four heteroatoms selected fromnitrogen, oxygen, and sulfur) having from 3 to 12 ring atoms perheterocycle. Illustrative examples of heteroaryl groups include thefollowing entities, in the form of properly bonded moieties:

The term “halogen” represents chlorine, fluorine, bromine, or iodine.The term “halo” represents chloro, fluoro, bromo, or iodo.

The term “oxo” represents a carbonyl oxygen. For example, a cyclopentylsubstituted with oxo is cyclopentanone.

Those skilled in the art will recognize that the species listed orillustrated above are not exhaustive, and that additional species withinthe scope of these defined terms may also be selected.

The term “substituted” means that the specified group or moiety bearsone or more substituents. The term “unsubstituted” means that thespecified group bears no substituents. The term “optionally substituted”means that the specified group is unsubstituted or substituted by one ormore substituents. Where the term “substituted” is used to describe astructural system, the substitution is meant to occur at anyvalency-allowed position on the system.

Any formula depicted herein is intended to represent a compound of thatstructural formula as well as certain variations or forms. For example,a formula given herein is intended to include a racemic form, or one ormore enantiomeric, diastereomeric, or geometric isomers, or a mixturethereof. Additionally, any formula given herein is intended to referalso to a hydrate, solvate, or polymorph of such a compound, or amixture thereof.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S,¹⁸F, ³⁶Cl, and ¹²⁵I, respectively. Such isotopically labelled compoundsare useful in metabolic studies (preferably with ¹⁴C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques[such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT)] including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or ¹¹C labeled compound may be particularly preferredfor PET or SPECT studies. PET and SPECT studies may be performed asdescribed, for example, by Brooks, D. J., “Positron Emission Tomographyand Single-Photon Emission Computed Tomography in Central Nervous SystemDrug Development,” NeuroRx 2005, 2(2), 226-236, and references citedtherein. Further, substitution with heavier isotopes such as deuterium(i.e., ²H) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements. Isotopically labeled compounds of thisinvention and prodrugs thereof can generally be prepared by carrying outthe procedures disclosed in the schemes or in the examples andpreparations described below by substituting a readily availableisotopically labeled reagent for a non-isotopically labeled reagent.

The nomenclature “C_(i-j)” with j>i, when applied herein to a class ofsubstituents, is meant to refer to embodiments of this invention forwhich each and every one of the number of carbon members, from i to jincluding i and j, is independently realized. By way of example, theterm C₁₋₃ refers independently to embodiments that have one carbonmember (C₁), embodiments that have two carbon members (C₂), andembodiments that have three carbon members (C₃).

Any disubstituent referred to herein is meant to encompass the variousattachment possibilities when more than one of such possibilities areallowed. For example, reference to disubstituent -A-B-, where A≠B,refers herein to such disubstituent with A attached to a firstsubstituted member and B attached to a second substituted member, and italso refers to such disubstituent with A attached to the secondsubstituted member and B attached to the first substituted member.

The invention also includes pharmaceutically acceptable salts of thecompounds represented by Formula (I), preferably of those describedabove and of the specific compounds exemplified herein, andpharmaceutical compositions comprising such salts, and methods of usingsuch salts.

A “pharmaceutically acceptable salt” is intended to mean a salt of afree acid or base of a compound represented herein that is non-toxic,biologically tolerable, or otherwise biologically suitable foradministration to the subject. See, generally, S. M. Berge, et al.,“Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66, 1-19. Preferredpharmaceutically acceptable salts are those that are pharmacologicallyeffective and suitable for contact with the tissues of subjects withoutundue toxicity, irritation, or allergic response. A compound describedherein may possess a sufficiently acidic group, a sufficiently basicgroup, both types of functional groups, or more than one of each type,and accordingly react with a number of inorganic or organic bases, andinorganic and organic acids, to form a pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogen-phosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,methylsulfonates, propylsulfonates, besylates, xylenesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates,phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists ofother suitable pharmaceutically acceptable salts are found inRemington's Pharmaceutical Sciences, 17th Edition, Mack PublishingCompany, Easton, Pa., 1985.

For a compound of Formula (I) that contains a basic nitrogen, apharmaceutically acceptable salt may be prepared by any suitable methodavailable in the art, for example, treatment of the free base with aninorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuricacid, sulfamic acid, nitric acid, boric acid, phosphoric acid, and thelike, or with an organic acid, such as acetic acid, phenylacetic acid,propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid,hydroxymaleic acid, isethionic acid, succinic acid, valeric acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, oleic acid, palmitic acid, lauric acid, a pyranosidylacid, such as glucuronic acid or galacturonic acid, an alpha-hydroxyacid, such as mandelic acid, citric acid, or tartaric acid, an aminoacid, such as aspartic acid or glutamic acid, an aromatic acid, such asbenzoic acid, 2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, asulfonic acid, such as laurylsulfonic acid, p-toluenesulfonic acid,methanesulfonic acid, or ethanesulfonic acid, or any compatible mixtureof acids such as those given as examples herein, and any other acid andmixture thereof that are regarded as equivalents or acceptablesubstitutes in light of the ordinary level of skill in this technology.

The invention also relates to pharmaceutically acceptable prodrugs ofthe compounds of Formula (I), and treatment methods employing suchpharmaceutically acceptable prodrugs. The term “prodrug” means aprecursor of a designated compound that, following administration to asubject, yields the compound in vivo via a chemical or physiologicalprocess such as solvolysis or enzymatic cleavage, or under physiologicalconditions (e.g., a prodrug on being brought to physiological pH isconverted to the compound of Formula (I)). A “pharmaceuticallyacceptable prodrug” is a prodrug that is non-toxic, biologicallytolerable, and otherwise biologically suitable for administration to thesubject. Illustrative procedures for the selection and preparation ofsuitable prodrug derivatives are described, for example, in “Design ofProdrugs”, ed. H. Bundgaard, Elsevier, 1985.

The present invention also relates to pharmaceutically activemetabolites of compounds of Formula (I), and uses of such metabolites inthe methods of the invention. A “pharmaceutically active metabolite”means a pharmacologically active product of metabolism in the body of acompound of Formula (I) or salt thereof. Prodrugs and active metabolitesof a compound may be determined using routine techniques known oravailable in the art. See, e.g., Bertolini et al., J. Med. Chem. 1997,40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86 (7), 765-767;Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984,13, 255-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); andLarsen, Design and Application of Prodrugs, Drug Design and Development(Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).

Pharmaceutical Compositions

For treatment purposes, pharmaceutical compositions comprising thecompounds described herein may further comprise one or morepharmaceutically-acceptable excipients. A pharmaceutically-acceptableexcipient is a substance that is non-toxic and otherwise biologicallysuitable for administration to a subject. Such excipients facilitateadministration of the compounds described herein and are compatible withthe active ingredient. Examples of pharmaceutically-acceptableexcipients include stabilizers, lubricants, surfactants, diluents,anti-oxidants, binders, coloring agents, bulking agents, emulsifiers, ortaste-modifying agents. In preferred embodiments, pharmaceuticalcompositions according to the invention are sterile compositions.Pharmaceutical compositions may be prepared using compounding techniquesknown or that become available to those skilled in the art.

Sterile compositions are also contemplated by the invention, includingcompositions that are in accord with national and local regulationsgoverning such compositions.

The pharmaceutical compositions and compounds described herein may beformulated as solutions, emulsions, suspensions, or dispersions insuitable pharmaceutical solvents or carriers, or as pills, tablets,lozenges, suppositories, sachets, dragees, granules, powders, powdersfor reconstitution, or capsules along with solid carriers according toconventional methods known in the art for preparation of various dosageforms. Pharmaceutical compositions of the invention may be administeredby a suitable route of delivery, such as oral, parenteral, rectal,nasal, topical, or ocular routes, or by inhalation. Preferably, thecompositions are formulated for intravenous or oral administration.

For oral administration, the compounds the invention may be provided ina solid form, such as a tablet or capsule, or as a solution, emulsion,or suspension. To prepare the oral compositions, the compounds of theinvention may be formulated to yield a dosage of, e.g., from about 0.01to about 50 mg/kg daily, or from about 0.05 to about 20 mg/kg daily, orfrom about 0.1 to about 10 mg/kg daily. Additional dosages include fromabout 0.1 mg to 1 g daily, from about 1 mg to about 10 mg daily, fromabout 10 mg to about 50 mg daily, from about 50 mg to about 250 mgdaily, or from about 250 mg to 1 g daily. Oral tablets may include theactive ingredient(s) mixed with compatible pharmaceutically acceptableexcipients such as diluents, disintegrating agents, binding agents,lubricating agents, sweetening agents, flavoring agents, coloring agentsand preservative agents. Suitable inert fillers include sodium andcalcium carbonate, sodium and calcium phosphate, lactose, starch, sugar,glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, andthe like. Exemplary liquid oral excipients include ethanol, glycerol,water, and the like. Starch, polyvinyl-pyrrolidone (PVP), sodium starchglycolate, microcrystalline cellulose, and alginic acid are exemplarydisintegrating agents. Binding agents may include starch and gelatin.The lubricating agent, if present, may be magnesium stearate, stearicacid, or talc. If desired, the tablets may be coated with a materialsuch as glyceryl monostearate or glyceryl distearate to delay absorptionin the gastrointestinal tract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules.To prepare hard gelatin capsules, active ingredient(s) may be mixed witha solid, semi-solid, or liquid diluent. Soft gelatin capsules may beprepared by mixing the active ingredient with water, an oil such aspeanut oil or olive oil, liquid paraffin, a mixture of mono anddi-glycerides of short chain fatty acids, polyethylene glycol 400, orpropylene glycol.

Liquids for oral administration may be in the form of suspensions,solutions, emulsions, or syrups, or may be lyophilized or presented as adry product for reconstitution with water or other suitable vehiclebefore use. Such liquid compositions may optionally contain:pharmaceutically-acceptable excipients such as suspending agents (forexample, sorbitol, methyl cellulose, sodium alginate, gelatin,hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel andthe like); non-aqueous vehicles, e.g., oil (for example, almond oil orfractionated coconut oil), propylene glycol, ethyl alcohol, or water;preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbicacid); wetting agents such as lecithin; and, if desired, flavoring orcoloring agents.

The inventive compositions may be formulated for rectal administrationas a suppository. For parenteral use, including intravenous,intramuscular, intraperitoneal, intranasal, or subcutaneous routes, theagents of the invention may be provided in sterile aqueous solutions orsuspensions, buffered to an appropriate pH and isotonicity or inparenterally acceptable oil. Suitable aqueous vehicles include Ringer'ssolution and isotonic sodium chloride. Such forms may be presented inunit-dose form such as ampoules or disposable injection devices, inmulti-dose forms such as vials from which the appropriate dose may bewithdrawn, or in a solid form or pre-concentrate that can be used toprepare an injectable formulation. Illustrative infusion doses rangefrom about 1 to 1000 μg/kg/minute of agent admixed with a pharmaceuticalcarrier over a period ranging from several minutes to several days.

For nasal, inhaled, or oral administration, the inventive pharmaceuticalcompositions may be administered using, for example, a spray formulationalso containing a suitable carrier.

For topical applications, the compounds of the present invention arepreferably formulated as creams or ointments or a similar vehiclesuitable for topical administration. For topical administration, theinventive compounds may be mixed with a pharmaceutical carrier at aconcentration of about 0.1% to about 10% of drug to vehicle. Anothermode of administering the agents of the invention may utilize a patchformulation to effect transdermal delivery.

As used herein, the terms “treat” or “treatment” encompass both“preventative” and “curative” treatment. “Preventative” treatment ismeant to indicate a postponement of development of a disease, a symptomof a disease, or medical condition, suppressing symptoms that mayappear, or reducing the risk of developing or recurrence of a disease orsymptom. “Curative” treatment includes reducing the severity of orsuppressing the worsening of an existing disease, symptom, or condition.Thus, treatment includes ameliorating or preventing the worsening ofexisting disease symptoms, preventing additional symptoms fromoccurring, ameliorating or preventing the underlying systemic causes ofsymptoms, inhibiting the disorder or disease, e.g., arresting thedevelopment of the disorder or disease, relieving the disorder ordisease, causing regression of the disorder or disease, relieving acondition caused by the disease or disorder, or stopping the symptoms ofthe disease or disorder.

The term “subject” refers to a mammalian patient in need of suchtreatment, such as a human.

Exemplary neurodegenerative diseases that are characterized by proteinaggregation include Alzheimer's Disease, Parkinson's Disease,fronto-temporal Dementia, Dementia with Lewy Bodies (Lewy body disease),Parkinson's Disease with Dementia, Multiple System Atrophy, AmyotrophicLateral Sclerosis, and Huntington's Disease, as well as cancers andinflammatory diseases.

In one aspect, the compounds and pharmaceutical compositions of theinvention specifically target α-synuclein, β-amyloid, and/or tau proteinaggregates. Thus, these compounds and pharmaceutical compositions can beused to prevent, reverse, slow, or inhibit aggregation of α-synuclein,β-amyloid, and/or tau proteins, and are used in methods of the inventionto treat degenerative neurological diseases related to or caused byaggregation, e.g., such as aggregation of α-synuclein, β-amyloid, and/ortau proteins. Preferably, the methods of the invention targetneurodegenerative diseases associated with aggregation of α-synuclein,β-amyloid, and/or tau protein. In preferred embodiments, methods oftreatment target Parkinson's disease, Alzheimer's disease, Lewy bodydisease, or multiple system atrophy. In other embodiments, the methodstarget cancer or melanoma. The compounds, compositions, and method ofthe present invention are also used to mitigate deleterious effects thatare secondary to protein aggregation, such as neuronal cell death.

In some aspects, the compounds, compositions, and methods of theinvention are used to target α-synuclein (SYN) aggregation. Inalternative aspects, the compounds, compositions, and methods of theinvention are used to target Aβ aggregation.

In the inhibitory methods of the invention, an “effective amount” meansan amount sufficient to reduce, slow the progression of, or reverseprotein or peptide aggregation. Measuring the amount of aggregation maybe performed by routine analytical methods such as those describedbelow. Such modulation is useful in a variety of settings, including invitro assays. In such methods, the cell is preferably a nerve cell.

In treatment methods according to the invention, an “effective amount”means an amount or dose sufficient to generally bring about the desiredtherapeutic benefit in subjects needing such treatment. Effectiveamounts or doses of the compounds of the invention may be ascertained byroutine methods, such as modeling, dose escalation, or clinical trials,taking into account routine factors, e.g., the mode or route ofadministration or drug delivery, the pharmacokinetics of the agent, theseverity and course of the infection, the subject's health status,condition, and weight, and the judgment of the treating physician. Anexemplary dose is in the range of about 1 ug to 2 mg of active agent perkilogram of subject's body weight per day, preferably about 0.05 to 100mg/kg/day, or about 1 to 35 mg/kg/day, or about 0.1 to 10 mg/kg/day. Inalternative embodiments an exemplary dose is in the range of about 1 mgto about 1 g per day, or about 1-500, 1-250, 1-100, 1-50, 50-500, or250-500 mg per day. The total dosage may be given in single or divideddosage units (e.g., BID, TID, QID).

Once improvement of the patient's disease has occurred, the dose may beadjusted for preventative or maintenance treatment. For example, thedosage or the frequency of administration, or both, may be reduced as afunction of the symptoms, to a level at which the desired therapeutic orprophylactic effect is maintained. Of course, if symptoms have beenalleviated to an appropriate level, treatment may cease. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of symptoms. Patients may also require chronic treatment on along-term basis.

Drug Combinations

The inventive compounds described herein may be used in pharmaceuticalcompositions or methods in combination with one or more additionalactive ingredients in the treatment of neurodegenerative disorders.Further additional active ingredients for cancer applications includeother cancer therapeutics or agents that mitigate adverse effects ofcancer chemotherapeutic agents. Such combinations may serve to increaseefficacy, ameliorate other disease symptoms, decrease one or more sideeffects, or decrease the required dose of an inventive compound. Theadditional active ingredients may be administered in a separatepharmaceutical composition from a compound of the present invention ormay be included with a compound of the present invention in a singlepharmaceutical composition. The additional active ingredients may beadministered simultaneously with, prior to, or after administration of acompound of the present invention.

Combination agents include additional active ingredients are those thatare known or discovered to be effective in treating neurodegenerativedisorders, including those active against another target associated withthe disease, such as but not limited to, a) compounds that addressprotein misfolding (such as drugs which reduce the production of theseproteins, which increase their clearance or which alter theiraggregation and/or propagation); b) compounds that treat symptoms ofsuch disorders (e.g., dopamine replacement therapies); and c) drugs thatact as neuroprotectants by complementary mechanisms (e.g., thosetargeting autophagy, those that are anti-oxidants, and those acting byother mechanisms such as adenosine A2A antagonists).

For example, compositions and formulations of the invention, as well asmethods of treatment, can further comprise other drugs orpharmaceuticals, e.g., other active agents useful for treating orpalliative for a degenerative neurological disease related to or causedby protein aggregation, e.g., synuclein, beta-amyloid and/or tau proteinaggregation, e.g., Parkinson's disease, Alzheimer's Disease (AD), Lewybody disease (LBD) and multiple system atrophy (MSA), or relatedsymptoms or conditions. For example, the pharmaceutical compositions ofthe invention may additional comprise one or more of such active agents,and methods of treatment may additionally comprise administering aneffective amount of one or more of such active agents. In certainembodiments, additional active agents may be antibiotics (e.g.,antibacterial or bacteriostatic peptides or proteins), e.g., thoseeffective against gram positive or negative bacteria, fluids, cytokines,immunoregulatory agents, anti-inflammatory agents, complement activatingagents, such as peptides or proteins comprising collagen-like domains orfibrinogen-like domains (e.g., a ficolin), carbohydrate-binding domains,and the like and combinations thereof. Additional active agents includethose useful in such compositions and methods include dopamine therapydrugs, catechol-O-methyl transferase (COMT) inhibitors, monamine oxidaseinhibitors, cognition enhancers (such as acetylcholinesterase inhibitorsor memantine), adenosine 2A receptor antagonists, beta-secretaseinhibitors, or gamma-secretase inhibitors. In particular embodiments, atleast one compound of the present invention may be combined in apharmaceutical composition or a method of treatment with one or moredrugs selected from the group consisting of: tacrine (Cognex), donepezil(Aricept), rivastigmine (Exelon) galantamine (Reminyl), physostigmine,neostigmine, Icopezil (CP-118954,5,7-dihydro-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo-[4,5-f-]-1,2-benzisoxazol-6-onemaleate), ER-127528(4-[(5,6-dimethoxy-2-fluoro-1-indanon)-2-yl]methyl-1-(3-fluorobenzyl)pipe-ridinehydrochloride), zanapezil (TAK-147;3-[1-(phenylmethyl)piperidin-4-yl]-1-(2,3,4,5-tetrahydro-1H-1-benzazepin-8-yl)-1-propanefumarate), Metrifonate (T-588;(−)-R-.alpha.-[[2-(dimethylamino)ethoxy]methyl]benzo[b]thiophene-5-methanolhydrochloride), FK-960(N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide-hydrate), TCH-346(N-methyl-N-2-pyropinyldibenz[b,f]oxepine-10-methanamine), SDZ-220-581((S)-alpha-amino-5-(phosphonomethyl)-[1,1′-biphenyl]-3-propionic acid),memantine (Namenda/Exiba) and 1,3,3,5,5-pentamethylcyclohexan-1-amine(Neramexane), tarenflurbil (Flurizan), tramiprosate (Alzhemed),clioquinol, PBT-2 (an 8-hydroxyquinilone derivative),1-(2-(2-Naphthyl)ethyl)-4-(3-trifluoromethylphenyl)-1,2,3,6-tetrahydropyridine,Huperzine A, posatirelin, leuprolide or derivatives thereof,ispronicline, (3-aminopropyl)(n-butyl)phosphinic acid (SGS-742),N-methyl-5-(3-(5-isopropoxypyridinyl))-4-penten-2-amine (ispronicline),1-decanaminium, N-(2-hydroxy-3-sulfopropyl)-N-methyl-N-octyl-, innersalt (zt-1), salicylates, aspirin, amoxiprin, benorilate, cholinemagnesium salicylate, diflunisal, faislamine, methyl salicylate,magnesium salicylate, salicyl salicylate, diclofenac, aceclofenac,acemetacin, bromfenac, etodolac, indometacin, nabumetone, sulindac,tolmetin, ibuprofen, carprofen, fenbufen, fenoprofen, flurbiprofen,ketoprofen, ketorolac, loxoprofen, naproxen, tiaprofenic acid, suprofen,mefenamic acid, meclofenamic acid, phenylbutazone, azapropazone,metamizole, oxyphenbutazone, sulfinprazone, piroxicam, lornoxicam,meloxicam, tenoxicam, celecoxib, etoricoxib, lumiracoxib, parecoxib,rofecoxib, valdecoxib, nimesulide, arylalkanoic acids, 2-arylpropionicacids (profens), N-arylanthranilic acids (fenamic acids), pyrazolidinederivatives, oxicams, COX-2 inhibitors, sulphonanilides, essential fattyacids, and Minozac(2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidinedihydrochloride hydrate), or a combination thereof.

Potential combination agents for cancer therapies may include, forexample, protein and lipid kinase inhibitors (e.g., PI3K, B-raf,BCR/ABL), radiation treatment enhancers, microtubule binders (e.g.,taxol, vinblastine), cell metabolism inhibitors, DNA intercalators,topoisomerase inhibitors (e.g., doxorubicin), and DNA alkylating agents.

Assays

The compounds described herein can be used in research applications,including in in vitro, in vivo, or ex vivo experimental systems.Experimental systems can include, without limitation, cell samples,tissue samples, cell components or mixtures of cell components, whole orpartial organs, or organisms. Research applications include, withoutlimitation, use as assay reagents, elucidation of biochemical pathways,or evaluation of the effects of other agents on the experimental systemin the presence or absence of one or more compounds described herein.

The compounds described herein can also be used in biochemical assays.In some embodiments, a compound described herein can be incubated with atissue or cell sample from a subject to evaluate the subject's potentialresponse to administration of the compound, or to determine whichcompound described herein produces the optimum effect in a specificsubject or set of subjects. One such assay would involve (a) obtaining acell sample or tissue sample from a subject in which modulation of oneor more biomarkers can be assayed; (b) administering one or morecompounds described herein to the cell sample or tissue sample; and (c)determining the amount of modulation of the one or more biomarkers afteradministration of the compound, compared to the status of the biomarkerprior to administration of the compound. Optionally, following step (c),the assay would involve an additional step (d) selecting a compound foruse in treating a disease or medical condition associated with proteinaggregation based on the amount of modulation determined in step (c).

Chemical Synthesis

Exemplary chemical entities useful in methods of the invention will nowbe described by reference to illustrative synthetic schemes for theirgeneral preparation below and the specific examples that follow.Artisans will recognize that, to obtain the various compounds herein,starting materials may be suitably selected so that the ultimatelydesired substituents will be carried through the reaction scheme with orwithout protection as appropriate to yield the desired product.Alternatively, it may be necessary or desirable to employ, in the placeof the ultimately desired substituent, a suitable group that may becarried through the reaction scheme and replaced as appropriate with thedesired substituent. Furthermore, one of skill in the art will recognizethat the transformations shown in the schemes below may be performed inany order that is compatible with the functionality of the particularpendant groups. Each of the reactions depicted in the general schemes ispreferably run at a temperature from about 0° C. to the refluxtemperature of the organic solvent used. Unless otherwise specified, thevariables are as defined above in reference to Formula (I). Isotopicallylabeled compounds as described herein are prepared according to themethods described below, using suitably labeled starting materials. Suchmaterials are generally available from commercial suppliers ofradiolabeled chemical reagents.

Compounds of Formula (I) are prepared as shown in Scheme A Amino-ethylindole derivatives A1 are commercially available or are preparedaccording to Scheme B. Compounds A1 are coupled with activated acylcompounds A2, wherein X is, for example, —OH or —Cl, under standardamide formation conditions to produce compounds of Formula (I).

As shown in Scheme B, substituted indoles A1 are prepared frommethyl-indoles B1 by acylation followed by reductive amination.

Heterocyclic compounds C4 are prepared according to Scheme C. Certaincompounds A, C1, A-CO₂R (where R is H or C₁₋₄alkyl), and C2 arecommercially available. In some embodiments, heterocycles A arehalogenated to form halo compounds C1, and then are acylated to formbis-functionalized compounds C2. In other embodiments, compounds A-CO₂Rare halogenated to form compounds C2. Coupling with amines HNR³R⁴ understandard amide coupling conditions provides compounds C3. Hydrolysis ofesters C3 yields amino acids C4, which can be used in coupling reactionsas shown in Scheme A.

As shown in Scheme D, methyl-heterocyclic compounds D1 are homologatedwith, for example, paraformaldehyde, to provide hydroxyethyl compoundsD2. Activation of the hydroxyl group as, for example, a halide ortosylate, and displacement with HNR³R⁴, yields amino compounds D3.Acylation of the heterocyclic ring gives esters D4, and hydrolysisgenerates amino acids D5.

EXAMPLES

The following examples are offered to illustrate but not to limit theinvention. One of skill in the art will recognize that the followingsynthetic reactions and schemes may be modified by choice of suitablestarting materials and reagents in order to access other compounds ofFormula (I).

Example 1N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide

Step 1.

To a solution of compound 1A (6 g, 45.8 mmol) in dry 1,2-dichloroethane(80 mL) was added AlCl₃ (18.3 g, 137.4 mmol) at 0° C. The mixture waswarmed to 25° C. and stirred for 30 min. The mixture was cooled to 0°C., and compound 1B (6.2 g, 51.3 mmol) was added dropwise. The mixturewas stirred at 25° C. for 48 h. The reaction mixture was poured into icewater slowly and extracted with dichloromethane (DCM, three times (3×)).The organic layer was washed with brine (3×), dried over Na₂SO₄ andconcentrated. The residue was purified by column (20:1 petroleumether/EtOAc) to give compound 1C (4.8 g, 49%) as a yellow solid. ¹H NMR(400 MHz, CDCl₃) δ 8.16 (br s, 1H), 7.39 (d, J=8.0 Hz, 1H), 7.37 (d,J=8.0 Hz, 1H), 7.23 (t, J=7.2 Hz, 1H), 7.15 (t, J=7.2 Hz, 1H), 7.15 (s,1H), 3.83 (s, 2H), 2.50 (t, J=8.8 Hz, 2H), 1.54-1.63 (m, 2H), 1.24-1.29(m, 2H), 0.86 (t, J=7.6 Hz, 3H).

Step 2.

A mixture of compound 1C (4.8 g, 22.3 mmol) in MeOH (150 mL) was addedAcONH₄ (3.35 g, 44.6 mmol) and NaBH₃CN (2.8 g, 44.6 mmol). The mixturewas refluxed for 24 h. The mixture was concentrated, and the residuedissolved in DCM (150 mL), washed with brine (3×), dried over Na₂SO₄,and concentrated to give crude compound 1D (2.4 g, 50%) as a yellowsolid. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (s, 1H), 7.54 (d, J=8.0 Hz, 1H),7.29 (d, J=8.0 Hz, 1H), 7.03-7.18 (m, 2H), 6.98 (s, 1H), 3.01-3.03 (m,1H), 2.89 (dd, J=14.0, 4.0 Hz, 1H), 2.52 (dd, J=14.0, 8.8 Hz, 1H),1.18-1.50 (m, 8H), 0.85 (t, J=7.2 Hz, 3H).

Step 3.

A mixture of compound 1E (2.2 g, 10.0 mmol), N-methylpiperazine (1.1 g,11.0 mmol), and K₂CO₃ (3.4 g, 24.9 mmol) in acetonitrile (70 mL) wasstirred at 80° C. for 24 h. The mixture was concentrated and dilutedwith H₂O and extracted with EtOAc (3×). The combined organic layers weredried and then concentrated to give compound 1G (2.5 g, >100%) as abrown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H), 4.28 (q, J=7.2 Hz,2H), 3.58 (t, J=5.6 Hz, 4H), 2.50 (t, J=5.6 Hz, 4H), 2.33 (s, 3H), 1.32(t, J=7.2 Hz, 3H).

Step 4.

A mixture of compound 1G (2.0 g, 8.3 mmol) in THF (20 mL) was addedsolution of NaOH (1.33 g, 33.2 mmol) in H₂O (40 mL). The mixture wasstirred for 24 h at 80° C. The mixture was concentrated to remove THFand extracted with n-BuOH. The organic layer was dried and concentratedto give compound 1H (1.38 g, 66.7%) as a white solid. ¹H NMR (400 MHz,MeOD) δ 7.55 (s, 1H), 3.49-3.52 (m, 4H), 2.53-2.56 (m, 4H), 2.36 (s,3H).

Step 5. To a mixture of compound 1H (1.1 g, 5.1 mmol) in CH₂Cl₂ (50 mL)and THF (5 mL) was added compound 1D (2 g, 8.09 mmol), followed bybenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP, 3.18 g, 6.12 mmol) and DIPEA (2.6 g, 20.4 mmol). After 24 h at25° C. under N₂, the mixture was diluted with H₂O (40 mL) and extractedwith DCM (3×). The combined organic layers were dried over Na₂SO₄,concentrated, and the residue purified by preparative-HPLC (ShimadzuLC-8A Preparative HPLC, Luna(2) C18 column, 26%-56% acetonitrile inNH₄OAc over 20 min at 80 mL/min) to give Example 1 (576 mg, 27%) as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.39 (1H, s), 7.38(d, J=7.2 Hz, 1H), 7.22 (d, J=7.2 Hz, 1H), 7.07-7.21 (m, 2H), 7.06 (s,1H), 5.50 (d, J=8.4 Hz, 1H), 4.40-4.46 (m, 1H), 3.56 (t, J=5.2 Hz, 4H),3.0-3.1 (m, 2H), 2.52 (t, J=4.8 Hz, 4H), 2.36 (s, 3H), 1.26-1.60 (m,6H), 0.88 (t, J=7.2 Hz, 3H). LCMS: 100% (M+1⁺): 426.2.

Alternate Synthesis

To a mixture of compound 3A (400 g, 0.98 mol, see below) and K₂CO₃ (340g, 2.5 mol) in CH₃CN (3 L) was added compound 1F (197 g, 1.97 mol). Themixture was stirred for 12 h at 80° C. under N₂ atmosphere. The mixturewas diluted with water (4 L) and extracted with DCM (4 L×3). Thecombined organic layers were dried over Na₂SO₄ and concentrated. Theresidue was washed with 1:1 petroleum ether/ethyl acetate to giveExample 1 (200 g, 44%) as a white solid. 4 M HCl in ethyl acetate (235mL) was added to a solution of Example 1 (200 g, 0.47 mol) in DCM (2 L).The mixture was stirred for 1 h at room temperature, the solvent wasconcentrated, and the residue was recrystallized from methyl t-butylether (1 L). The resultant solid was collected by filtration and driedunder reduced pressure at 50° C. to give the HCl salt of Example 1 (210g, 92%).

Example 2N-(2-(1H-Indol-3-yl)ethyl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide

Step 1.

A solution of NaOH (4.1 g, 102 mmol) in H₂O (100 mL) was added dropwiseto a solution of compound 2A (20 g, 84.7 mmol) in tetrahydrofuran (THF,100 mL) at 0° C. The mixture was stirred at 10° C. for 1 h. The mixturewas neutralized with HCl (6 M) and extracted with EtOAc (3×). Theorganic layer was washed by brine, dried over Na₂SO₄, and concentratedto give compound 2B (17.7 g, 100%) as a yellow solid. ¹H NMR (400 MHz,DMSO-d₆) δ 8.21 (s, 1H).

Step 2.

1,1-Carbonyl-diimidazole (2.06 g, 12.75 mmol) was added to a mixture ofcompound 2B (2.6 g, 12.5 mmol) in THF (16 mL). The mixture was stirredfor 1 h at room temperature, and then was treated with triethylamine(TEA, 2.53 g, 25 mmol) and compound 2C (2 g, 12.5 mmol). The resultingmixture was stirred at room temperature for 12 h. The mixture wasdiluted with EtOAc, washed with 1M HCl (2×) and brine, then dried overNa₂SO₄, and concentrated to give compound 2D (4.41 g, >100%) as a yellowsolid.

Step 3. To a mixture of compound 2D (1 g, 5.7 mmol) and K₂CO₃ (1.0 g,7.2 mmol) in CH₃CN (6 mL) was added N-methylpiperazine (0.6 g, 5.7mmol). The mixture was stirred for 12 h at 80° C. under N₂ atmosphere.Water was added and the mixture was extracted with DCM (5×). Thecombined organic layers were dried over Na₂SO₄ and concentrated to givea yellow solid (0.8 g), which was washed with methyl tertiary butylether (5 mL) to give Example 2 (0.3 g, 29%) as a white solid. ¹H NMR(400 MHz, DMSO-d₆) δ 8.37 (m, 1H), 7.78 (s, 1H), 7.56 (d, J=7.6 Hz, 1H),7.40 (d, J=8.4 Hz, 1H), 7.38-7.35 (m, 2H), 7.16 (s, 1H), 7.07 (t, J=7.4Hz, 1H), 6.98 (t, J=7.4 Hz, 1H), 3.48-3.44 (m, 6H), 2.90 (t, J=7.2 Hz,2H), 2.42-2.39 (m, 2H), 2.22 (s, 3H). MS: (M+H⁺): 370.

Example 3N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(piperazin-1-yl)thiazole-5-carboxamide

Step 1.

1,1-Carbonyl-diimidazole (4.04 g, 24.96 mmol) was added to a mixture ofcompound 2B (5.19 g, 24.96 mmol) in THF (40 mL). The mixture was stirredfor 1 h at room temperature and then was treated with TEA (7.56 g, 74.88mmol) and compound 1D (5.4 g, 24.96 mmol). After 12 h at roomtemperature, the mixture was diluted with EtOAc, washed successivelywith 1 M HCl (2×) and brine, dried over Na₂SO₄, and concentrated to givecompound 3A (7.74 g, 76%) as a yellow solid. ¹H NMR (400 MHz, MeOD-d₄) δ8.36 (d, 1H, J=8.4 Hz), 7.97 (s, 1H), 7.56 (d, J=8 Hz, 1H), 7.30 (d, J=8Hz, 1H), 7.06 (t, J=7.2 Hz, 1H), 7.05 (s, 1H), 6.95 (t, J=7.2 Hz, 1H),4.31-4.28 (m, 1H), 3.0-2.97 (m, 1H), 1.75-1.50 (m, 2H), 1.45-1.25 (m,4H), 2.22 (t, J=6.8 Hz, 3H). MS: (M+H⁺): 406, 408.

Step 2.

To a mixture of compound 3A (2 g, 5.7 mmol) and K₂CO₃ (1.77 g, 12.8mmol) in CH₃CN (12 mL) was added compound 3B (0.92 g, 4.9 mmol). After12 h at 80° C. under N₂ atmosphere, water was added (30 mL) and themixture was extracted with EtOAc (3×). The combined organic layers weredried over Na₂SO₄, concentrated, and purified by column chromatography(10 to 67% EtOAc/petroleum ether) to give compound 3C (2 g, 79%) as ayellow solid. This compound was used directly in the next step withoutcharacterization.

Step 3. A solution of compound 3C (1 g, 2.0 mmol) in methanol (10 mL)was treated with HCl (10 mL, 4 M in methanol). After stirring at roomtemperature for 3 h, the solvent was removed and the residue was pouredinto ice water (20 mL), adjusted pH to 9 with NaHCO₃, and extracted withEtOAc (2×). The combined organic layers were dried over Na₂SO₄ andconcentrated to give a yellow solid (0.45 g). Purification by columnchromatography (petroleum ether/DCM/MeOH 50/50/0 to 0/90/10) gaveExample 3 (0.32 g, 39%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.16(s, 1H), 7.64 (d, J=7.6 Hz, 1H), 7.39-7.36 (m, 2H), 7.18 (t, J=7.6 Hz,1H), 7.13 (t, J=7.6 Hz, 1H), 7.05 (s, 1H), 5.53 (d, J=8.8 Hz, 1H),4.46-4.38 (m, 1H), 3.58-3.49 (m, 4H), 3.06-2.96 (m, 6H), 2.05 (s, 1H),1.65-1.60 (m, 1H), 1.48-1.32 (m, 5H), 0.90-0.86 (m, 3H). MS: (M+H⁺):412.

Example 4N-(2-(1H-Indol-3-yl)ethyl)-2-(2-(4-methylpiperazin-1-yl)ethyl)thiazole-5-carboxamide

Step 1.

A mixture of compound 4A (50 g, 0.5 mol) and paraformaldehyde (50 g) ina sealed tube was stirred at 140° C. for 3 h. The reaction mixture waspurified by column chromatography (50% DCM/EtOAc) to give compound 4B(27 g, 41%) as a light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (s,1H), 7.16 (s, 1H), 3.91-3.98 (m, 2H), 3.23-3.28 (m, 2H).

Step 2.

To a solution of compound 4B (27 g, 0.2 mmol) in THF (100 mL) was addeda solution of NaOH (16.7 g, 0.4 mol) in water (100 mL) at 0° C. Afterstirring for 10 min, 4-methylbenzene-1-sulfonyl chloride (59.5 g, 0.3mol) was added portionwise. The mixture was allowed to warm to roomtemperature and was stirred a total of 2 h. The mixture was extractedwith EtOAc (3×). The organic phase was washed with brine, dried overNa₂SO₄, concentrated, and purified by column chromatography(hexane/EtOAc=2:1) to give compound 4C (32 g, 54%) as a colorless oil.¹H NMR (400 MHz, CDCl₃) δ 7.75 (2H, d, J=8 Hz, 2H), 7.65 (1H, d, J=4 Hz,1H), 7.32 (d, J=8 Hz, 2H), 7.22 (d, J=4 Hz, 1H), 4.40 (t, J=8 Hz, 2H),3.80 (t, J=8 Hz, 2H), 2.45 (s, 3H).

Step 3.

To a solution of compound 4C (32 g, 0.11 mol) in acetonitrile (300 mL)was added N-methylpiperazine (16.9 g, 0.16 mol) and Cs₂CO₃ (55 g, 0.16mol). The mixture was stirred at 60° C. overnight, then the mixture wasfiltered and the filtrate was concentrated, the residue was purified bycolumn chromatography (DCM/MeOH=10:1) to give compound 4D (14 g, 47%) asa light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J=3.2 Hz, 1H),7.19 (d, J=2.4 Hz, 1H), 3.21 (t, J=8.0 Hz, 2H), 2.79 (t, J=8.0 Hz, 2H),2.59 (s, 4H), 2.50 (s, 4H), 2.31 (s, 3H).

Step 4.

To a solution of compound 4D (14 g, 66 mmol) in anhydrous THF (70 mL)was added n-BuLi (32 mL, 80 mmol) dropwise at −78° C. The mixture wasstirred at this temperature for 30 min, then methyl carbonochloridate(7.52 g, 80 mmol) was added dropwise to the solution at the sametemperature. The mixture was stirred for 3 h, warming to roomtemperature during that time. The mixture was diluted with saturatedaqueous NH₄Cl (50 mL), extracted with EtOAc, washed with brine, driedover Na₂SO₄, and concentrated to give compound 4E (14 g, 78%). Thiscrude product can be used directly in the next step without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H), 3.89 (s, 3H), 3.19(t, J=8.0 Hz, 2H), 2.77 (t, J=8.0 Hz, 2H), 2.58 (s, 4H), 2.49 (s, 4H),2.17 (s, 3H).

Step 5.

To a solution of compound 4E (14 g, 52 mmol) in MeOH (100 mL) was addeda solution of NaOH (3.12 mL, 78 mmol) in water (39 mL). After 3 h atroom temperature, the mixture was concentrated and washed with EtOAc (30mL). The aqueous phase was adjusted to pH=5-6 with 1 N HCl and extractedwith EtOAc (3×). The combined organic layers were washed with brine,dried over Na₂SO₄, and concentrated to give compound 4F as an orangesolid (12 g, 92%). This crude product can be used directly in the nextstep without further purification.

Step 6. A solution of compound 4F (0.8 g, 3.1 mmol) in DCM (2 mL) andTHF (10 mL) was treated with 2-chloro-1-methyl-pyridinium iodide(Mukaiyama reagent, 1.0 g, 3.8 mmol) and N,N-diisopropylethylamine(DIPEA, 1.5 g, 16 mmol) and then stirred for 10 min. The mixture wasthen treated with 2-(1H-indol-3-yl)ethanamine (0.5 g, 3.1 mmol) and themixture was stirred at 50° C. for 3 h. The resulting precipitate wasfiltered off and the filtrate was concentrated in vacuo. The residue wasdissolved in ethyl acetate, washed with water (10 mL) and brine (10 mL),dried over Na₂SO₄, and concentrated. The residue was purified bypreparative HPLC (Shimadzu LC-8A, Gemini C-18, 12-42% CH₃CN in 0.04%aqueous NH₄OH over 20 min at 80 mL/min) to give Example 4 (200 mg, 16%)as a light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (s, 1H), 7.81(1H, s, 1H), 7.65 (d, J=8 Hz, 1H), 7.40 (d, J=8 Hz, 1H), 7.21 (t, J=8Hz, 1H), 7.08 (s, 1H), 5.99 (br s, 1H), 3.77 (q, J=6.2 Hz, 2H), 3.16 (t,J=7.1 Hz, 3H), 3.10 (t, J=6.6 Hz, 2H), 2.75 (t, J=6.8 Hz, 2H), 2.58 (brs, 4H), 2.49 (br s, 4H), 2.32 (s, 3H).

Example 5N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(2-(4-methylpiperazin-1-yl)ethyl)thiazole-5-carboxamide

A solution of compound 4F (1.2 g, 5 mmol) in DCM (2 mL) and THF (10 mL)was treated with Mukaiyama reagent (1.65 g, 6.5 mmol) and DIPEA (1.9 g,20 mmol) and then stirred for 10 min. The mixture was then treated withcompound 1D (1.2 g, 5 mmol) and was stirred at 50° C. for 3 h. Theresulting precipitate was filtered off and the filtrate was concentratedin vacuo. The residue was dissolved in ethyl acetate (30 mL), washedwith water (10 mL) and brine (10 mL), dried over Na₂SO₄, andconcentrated. Purification by preparative HPLC (Shimadzu LC-8A, GeminiC-18, 25-55% CH₃CN in 0.04% aqueous NH₄OH over 20 min at 80 mL/min) gaveExample 5 (250 mg, 14%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.10(s, 1H), 7.75 (s, 1H), 7.64 (d, J=8 Hz, 1H), 7.38 (d, J=8 Hz, 1H),7.19-7.21 (m, 1H), 7.13-7.14 (m, 1H), 5.13 (br s, 1H), 4.41-4.45 (m,1H), 3.13-3.18 (m, 1H), 3.05-3.09 (m, 1H), 2.74-2.77 (m, 1H), 2.58 (brs, 4H), 2.49 (br s, 4H), 2.32 (s, 3H), 1.63-1.70 (m, 1H), 1.49-1.54 (m,1H), 1.34-1.39 (m, 4H), 0.89 (t, J=4 Hz, 3H).

Example 6N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)oxazole-5-carboxamide

Step 1.

To a solution of compound 6A (15.0 g, 106 mmol) in THF (150 mL) wasadded dropwise lithium hexamethyl disilazide (178 mL, 170 mmol) at −60°C. The solution was stirred at −50° C. for 1 h. Then hexachloroethane(37.8 g, 160 mmol) was added to the solution. The solution was stirredat room temperature for 12 h. The reaction was quenched by saturated aq.NH₄Cl solution (50 mL) and extracted with EtOAc (50 mL). The organiclayer was separated, dried over Na₂SO₄, concentrated under vacuum andthe residue was purified with column chromatography to give compound 6B(10 g, 56%) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (s, 1H),3.39 (q, J=7.2 Hz, 2H), 1.38 (t, J=7.2 Hz, 3H).

Step 2.

A mixture of compound 6B (5.5 g, 31.3 mmol), N-ethylpiperazine (9.4 g,94 mmol), and K₂CO₃ (17.3 g, 125.2 mmol) in acetonitrile (80 mL) washeated at reflux at 80° C. for 2 h. The mixture was diluted with H₂O andextracted with EtOAc. The organic layer was separated, dried overNa₂SO₄, and concentrated under vacuum to give compound 6C (7 g, 94%) asa brown oil. ¹H NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H), 4.32 (q, J=7.2 Hz,2H), 3.66 (t, J=4.8 Hz, 4H), 2.48 (q, J=4.8 Hz, 4H), 2.34 (s, 3H), 1.34(t, J=7.2 Hz, 3H).

Step 3.

A mixture of compound 6C (2.0 g, 8.4 mmol) and NaOH (0.33 g, 8.4 mmol)in THF (10 mL) and H₂O (10 mL) was stirred at room temperature for 2 h.The mixture was concentrated under vacuum to give compound 6D (3.0 g,crude) as a yellow solid. ¹H NMR (400 MHz, D₂O) δ 7.26 (s, 1H), 3.51 (s,4H), 2.49 (s, 4H), 2.23 (s, 3H).

Step 4. A mixture of compound 6D (2.0 g, 9.5 mmol), compound 1D (1.64 g,7.6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 3.63 g,19 mmol), 1-hydroxy-benzotriazole (HOBt, 2.57 g, 19 mmol), and TEA (1.92g, 19 mmol) in DMF (30 mL) was stirred at room temperature for 12 h. Themixture was diluted with water and extracted with EtOAc. The organiclayer was separated, washed with water (3×), brine, dried over Na₂SO₄,and concentrated under vacuum. The residue was purified with columnchromatography (2 to 10% MeOH/DCM) to give Example 6 (220 mg, 6%) as awhite solid. ¹H NMR (400 MHz CDCl₃) δ 8.16 (s, 1H), 7.63 (d, J=8.0 Hz,1H), 7.40 (s, 1H), 7.39 (t, J=8.0 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 7.10(t, J=8.0 Hz, 1H), 7.08 (s, 1H), 5.69 (d, J=8.8 Hz, 1H), 4.47 (t, J=8.4Hz, 1H), 3.47 (d, J=2.0 Hz, 4H), 3.08-2.98 (m, 2H), 2.49 (t, J=4.4 Hz,4H), 2.36 (s, 3H), 1.66-1.51 (m, 1H), 1.51-1.49 (m, 1H), 1.39-1.27 (m,4H), 0.90 (d, J=7.2 Hz, 3H).

Example 7N-(1-(1H-Indol-3-yl)hexan-2-yl)-5-(4-methylpiperazin-1-yl)-1,3,4-thiadiazole-2-carboxamide

Step 1.

A solution of compound 7A (60 g, 0.313 mol), K₂CO₃ (130 g, 0.94 mol),and methyl piperazine in DMF (300 mL) was stirred at 40° C. for 3 h. Thereaction mixture was poured into water and extracted with CH₂Cl₂. Theorganic layer was washed with water, dried over Na₂SO₄, and concentratedto give compound 7B (58.5 g, 73%) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 4.37-4.47 (m, 2H), 3.60-3.71 (m, 4H), 2.47-2.60 (m, 4H), 2.34(s, 3H), 1.35-1.47 (m, 3H).

Step 2.

To a solution of compound 7B (5.0 g, 19.53 mmol) in THF (30 mL) wasadded 1 N aq. NaOH (30 mL) at room temperature and the mixture wasstirred for 3 h. The mixture was concentrated to remove THF, adjusted topH 8 with 1 N aq. HCl, and then freeze-dried to give crude the acid 7C(5.25 g, 100%) as a yellow solid (including NaCl), which was used fornext step without any purification. ¹H NMR (400 MHz, MeOD) δ 3.68 (m,4H), 2.91 (m, 4H), 2.57 (s, 3H).

Step 3. To a solution of compound 7C (450 mg, 2 mmol) in DMF (15 mL) andDCM (5 mL) was added EDC (400 mg, 2 mmol) and HOBt (310 mg, 2 mol) at 0°C. The mixture was stirred at 0° C. for 30 min. Compound 1D (500 mg, 2mmol) was added at 0° C. The mixture was stirred overnight at 40° C. Themixture was diluted with H₂O and extracted with EtOAc. The organic layerwas dried over Na₂SO₄ and concentrated to give crude product which waspurified by column chromatography (petroleum ether/DCM/MeOH 50/50/0 to0/10/1) and recrystallization (MeOH) to give Example 7 (230 g, 15%, 2batches) as an off-yellow solid. ¹H NMR: (400 MHz, DMSO-d₆) δ 10.74 (s,1H), 8.61 (d, J=8.0 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.30 (d, J=8.0 Hz,1H), 7.09 (s, 1H), 7.05-7.02 (m, 1H), 6.96-6.93 (m, 1H), 4.14 (s, 1H),3.50 (s, 4H), 2.99-2.84 (m, 2H), 2.42 (s, 4H), 2.21 (s, 3H), 1.57 (s,2H), 1.25-1.21 (m, 4H), 0.80 (s, 3H).

Example 8N-(1-(1H-Indol-3-yl)hexan-2-yl)-5-(4-methylpiperazin-1-yl)-4H-1,2,4-triazole-3-carboxamide

Step 1.

To a stirred mixture of compound 8A (4.5 g, 35 mmol) in 1 M sulfuricacid (70 mL, 70 mmol) was added a solution of sodium nitrite (2.41 g,52.5 mmol) in water (20 mL) dropwise at 0° C., followed by additionalwater (35 mL). After 25 min, a solution of KBr (8.33 g, 70 mmol) andcopper(I) bromide (4.51 g, 10.5 mmol) in water (35 mL) was added. Theresulting mixture was stirred at 20° C. for 3 h and the mixture wasextracted with ethyl acetate (3×) and the combined extracts washed withbrine (30 mL), dried over Na₂SO₄, and concentrated to dryness to givecompound 8B (2.9 g, 43%) as a white solid. ES-API Found: 191.9, 189.9.

Step 2.

To a solution of compound 8B (2.9 g, 15 mmol) in a mixture of DCM (5 mL)and THF (15 mL) was added Mukaiyama reagent (5 g, 19.5 mmol) and DIPEA(5.8 mL). After stirring for 10 min, 1-(1H-indol-3-yl)hexan-2-amine 1D(3.3 g, 15 mmol) was added to the mixture. The mixture was stirred at50° C. for 3 h. The mixture was diluted with DCM and washed with water.The organic phase was dried over Na₂SO₄ and concentrated, and theresidue was purified by column chromatography on silica gel (20:1DCM/MeOH) to give compound 8C (2.7 g, 47%) as a light yellow solid. ¹HNMR (400 MHz, CDCl₃) δ 7.96 (br s, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.23 (d,J=8.0 Hz, 1H), 7.07-7.11 (m, 2H), 7.01-7.04 (m, 1H), 6.96 (br s, 1H),4.38 (d, J=4 Hz, 1H), 2.95 (d, J=4 Hz, 1H), 1.61 (br s, 1H), 1.39-1.46(m, 1H), 1.19-1.28 (m, 4H), 0.78 (s, 3H).

Step 3. A mixture of compound 8C (778 mg, 2 mmol) and N-methylpiperazine(1 g, 10 mmol) was stirred in a sealed tube at 120° C. overnight. Ethylacetate (20 mL) was added and the precipitated solid was filtered off.The filtrate was washed with water and brine. The solution was driedover Na₂SO₄, concentrated, and purified by preparative TLC (10:1DCM/MeOH) to give Example 8 (184 mg, 18%) as a yellow solid. ¹H NMR (400MHz, CDCl₃) δ 8.43 (br s, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.26 (d, J=8.0Hz, 1H), 7.19 (s, 1H), 7.05 (t, J=8.0 Hz, 1H), 6.96-7.00 (m, 3H), 4.32(d, J=4 Hz, 1H), 3.42 (s, 4H), 2.28-2.29 (m, 2H), 2.50 (s, 4H), 2.28 (s,3H), 1.56-1.62 (m, 1H), 1.41-1.46 (m, 1H), 1.22-1.33 (m, 4H), 0.79 (t,J=8.0 Hz, 3H).

Example 9N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)-1H-imidazole-5-carboxamide

Step 1.

To a solution of imidazole 9A (20 g, 294 mmol) in THF (200 mL) and TEA(40 g, 400 mmol) was added dimethylsulfamoyl chloride (55 g, 383 mmol)slowly at 0° C. The mixture was stirred at room temperature overnight.The reaction mixture was poured into 300 mL of water and extracted withethyl acetate (3×). The solution was washed with water and brine, driedover Na₂SO₄, then concentrated to dryness to give compound 9B (42 g,89%) as a colorless oil, which solidified after standing at roomtemperature for 1 h. The solid material was used directly in the nextstep without further purification. ¹H NMR (400 MHz, CDCl₃) δ 7.86 (s,1H), 7.23 (s, 1H), 7.11 (s, 1H), 2.82 (s, 6H).

Step 2.

To a solution of compound 9B (10 g, 57 mmol) in anhydrous THF (100 mL)was added n-BuLi (27.3 mL, 68 mmol) dropwise at −78° C. The solution wasstirred at this temperature for 30 min. Perbromomethane (20.5 g, 62.7mmol) was then added at −78° C. and the mixture was allowed to rise toroom temperature over a period of 3 h followed by continued stirring atroom temperature (25° C.) overnight. The mixture was diluted with satd.aq. NH₄Cl (50 mL) and extracted with DCM (3×). The combined organiclayers were washed with brine, dried over Na₂SO₄, and concentrated, andthe residue was purified by MPLC (hexane/DCM 1:1) to give compound 9C(8.4 g, 57%) as a light oil. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (s, 1H),7.00 (s, 1H), 3.01 (s, 6H).

Step 3.

A mixture of compound 9C (10 g, 39 mmol) and N-methylpiperazine (12 g,118 mmol) in dioxane (100 mL) was stirred at 90° C. overnight. Thesolution was poured into water and extracted with DCM (3×). The combinedorganic layers were dried over Na₂SO₄ and concentrated and the residuewas purified by column chromatography (10:1 DCM/MeOH 10:1) to givecompound 9D (3.6 g, 34%) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ7.27 (s, 1H), 6.84 (s, 1H), 3.43 (m, 8H), 2.97 (s, 3H), 2.91 (s, 6H).

Step 4.

To a solution of compound 9D (3.6 g, 13 mmol) in anhydrous THF (40 mL)was added n-BuLi (6.3 mL, 16 mmol) dropwise at −78° C. The solution wasstirred at this temperature for 30 min, and then methylcarbonochloridate (1.48 g, 16 mmol) was added. The mixture was stirredfor 3 h at −78° C., and then was allowed to warm to room temperature.The mixture was diluted with satd. aq. NH₄Cl (50 mL) and extracted withDCM (3×). The combined organic layers were dried over Na₂SO₄ andconcentrated and the residue was purified by column chromatography (60:1DCM/MeOH) to give compound 9E (1.5 g, 54%) as a brown thick oil. ¹H NMR(400 MHz, CDCl₃) δ 7.29 (s, 1H), 3.76 (s, 3H), 3.50 (m, 4H), 2.77 (s,6H), 2.51 (m, 4H), 2.28 (s, 3H).

Step 5.

A mixture of compound 9E (1.5 g, 4.5 mmol) and concentrated HCl (7.5 mL)was stirred at 60° C. overnight. The mixture was concentrated undervacuum and the residue was treated with MeOH (10 mL). The white solidthat precipitated was collected by filtration and dried under vacuum togive compound 9F (800 mg, 84%) as a hydrochloride salt. ¹H NMR (400 MHz,D₂O) δ 7.53 (s, 1H), 4.09 (d, J=14 Hz, 2H), 3.67 (t, J=12.4 Hz, 2H),3.59 (d, J=12.4 Hz, 2H), 3.31 (t, J=12.4 Hz, 2H), 2.97 (s, 3H).

Step 6. To the solution of compound 9F (1.05 g, 5 mmol) in DCM (2 mL)and THF (10 mL) was added Mukaiyama reagent (1.65 g, 6.5 mmol) and DIPEA(1.9 g, 20 mmol). After stirring for 10 min,1-(1H-indol-3-yl)hexan-2-amine 1D (1.1 g, 5 mmol) was added and themixture was stirred at 50° C. for 3 h. The solvent was removed undervacuum and the residue was diluted with ethyl acetate and washed withwater and brine, dried over Na₂SO₄, and concentrated. The residue waspurified twice by preparative TLC (10:1 DCM/MeOH) to give Example 9 (200mg, 8.3%) as a light yellow solid. ¹H NMR (400 MHz, MeOD-d₄) δ 7.59 (d,J=8 Hz, 1H), 7.30 (d, J=8 Hz, 1H), 7.29 (s, 1H), 7.08-7.04 (m, 1H), 7.07(s, 1H), 6.98-6.94 (m, 1H), 4.33-4.26 (m, 1H), 3.40-3.35 (m, 4H),2.99-2.97 (m, 2H), 2.70-2.68 (m, 4H), 2.44 (s, 3H), 1.68-1.60 (m, 1H),1.55-1.45 (m, 1H) 1.38-1.28 (m, 4H), 0.86 (t, J=7 Hz, 3H).

Example 10N-(1-(5-Fluoro-1H-indol-3-yl)hexan-2-yl)-2-(2-morpholinoethyl)thiazole-5-carboxamide

Step 1.

A solution of compound 4C (12.7 g, 44.8 mmol) in acetonitrile (127 mL)was treated with morpholine (5.6 g, 65 mmol) and Cs₂CO₃ (21.3 g, 65mmol) and was stirred at 50° C. overnight. The mixture was filtered, thefiltrate was concentrated, and the residue was purified by columnchromatography (CH₂Cl₂/MeOH=10:1) to give compound 10A (5.6 g, 63%) as alight yellow oil. ¹H NMR (400 MHz, MeOD) δ 7.66 (d, J=3.2 Hz, 1H), 7.19(d, J=3.6 Hz, 1H), 3.72 (m, 4H), 3.22 (m, 2H), 2.77 (m, 2H), 2.52 (m,4H).

Step 2.

To a solution of compound 10A (5.6 g, 28 mmol) in anhydrous THF (56 mL)was added n-BuLi (13.6 mL, 17 mmol) dropwise at −78° C. The mixture wasstirred at this temperature for 30 min and then methyl carbonochloridate(3.2 g, 17 mmol) was added dropwise to the solution at −78° C. Themixture was stirred for 3 h. During this period, the temperature isallowed to rise up to room temperature (25° C.). Saturated NH₄Cl aqueoussolution (50 mL) was added to quench the reaction. The mixture wasextracted with EtOAc, washed with brine, dried over sodium sulfate andconcentrated to give compound 10B (4.0 g, 60%). This crude product wasused directly in the next step without further purification. ¹H NMR (400MHz, CDCl₃) δ 8.27 (s, 1H), 3.90 (s, 3H), 3.72-3.76 (m, 4H), 3.21 (t,J=8.0 Hz, 2H), 2.77 (t, J=8.0 Hz, 2H), 2.54 (s, 4H).

Step 3.

To a solution of compound 10B (3 g, 11.7 mmol) in MeOH (30 mL) was addedNaOH (0.7 g, 17 mmol) in water (9 mL). The mixture was stirred at 25° C.for 3 h. MeOH was removed in vacuo and the solution was washed withEtOAc (30 mL). The water phase was adjusted to pH=5-6 with 1 N HCl andextracted with EtOAc (50 mL×3). The organic phase was washed with brine,dried over sodium sulfate and concentrated to dryness to give compound10C as an orange solid (3 g, 100%). This crude product was used directlyin the next step without further purification.

Step 4.

To a solution of compound 10D (3.5 g, 18 mmol) in CH₂Cl₂ (35 mL) wasadded CDI (3.52 g, 21.8 mmol). The mixture was stirred at roomtemperature for 2 h and then N,O-dimethylhydroxylamine hydrochloride(2.3 g, 26 mmol) was added to the mixture. The mixture was stirred for 4h at room temperature. The mixture was diluted with water (30 mL) andextracted with EtOAc (50 mL×2). The organic phase was washed with brine,dried over sodium sulfate, concentrated to give compound 10E (4.5 g,100%) as brown oil. ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.15-7.30(m, 3H), 6.90-6.92 (m, 1H), 3.87 (s, 2H), 3.73 (s, 3H), 3.28 (s, 3H).

Step 5.

To a solution of compound 10E (3.57 g, 15 mmol) in anhydrous THF (72 mL)was added dropwise n-BuLi (46 mL, 91 mmol) at −78° C. The mixture wasstirred at this temperature for 10 min. Aqueous HCl (1 M, 30 mL) wasadded to quench the reaction. The mixture was extracted with EtOAc,washed with brine, dried over sodium sulfate and concentrated to givecompound 10F (3.5 g, 70%). This crude product was used directly in thenext step without further purification.

Step 6.

To a solution of AcONH₄ (46 g, 0.6 mol) and NaBH₃CN (9.5 g, 0.15 mol) inMeOH (140 mL) and THF (30 mL) was added compound 10F (3.5 g, 15 mmol).The mixture was stirred at room temperature for 20 h. MeOH and THF wereremoved in vacuo and sat. NaHCO₃ was added to the residue. The solutionwas extracted with EtOAc (100 mL×2). The organic phase was washed withbrine, dried over sodium sulfate and concentrated to give compound 10G(4.0 g, 100%) as brown oil. This crude product was used directly in thenext step without further purification.

Step 7. To a solution of compound 10C (1.6 g, 6.6 mmol) indichloromethane (6.4 mL) and THF (16 mL) was added Mukaiyama reagent(2.2 g, 8.6 mmol) and DIPEA (1.6 g, 6.6 mmol). The resultant mixture wasstirred for 10 min Compound 10G (1.6 g, 6.8 mmol) was added and themixture was stirred at 40° C. for 2 h. The resulting precipitate wasfiltered off and the filtrate was concentrated in vacuo. The residue wasdissolved in ethyl acetate (30 mL), washed with water (10 mL) and brine(10 mL), dried over sodium sulfate and concentrated. The residue waspurified by preparative HPLC (Shimadzu LC-8A Preparative HPLC, Luna(2)C18 column, 25%-55% acetonitrile in 10 mM aqueous NH₄HCO₃ over 20 min at80 mL/min) to give Example 10 (300 mg, 9.8%) as a white solid. ¹H NMR(400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.74 (s, 1H), 7.16 (t, J=4.4 Hz, 1H),7.01 (s, 1H), 6.81-6.86 (m, 1H), 5.80 (d, J=8.8 Hz, 1H), 4.33 (d, J=5.2Hz, 1H), 3.63 (s, 4H), 3.05 (t, J=6.4 Hz, 2H), 2.89-2.94 (m, 2H),2.64-2.66 (m, 2H), 2.43 (s, 4H), 1.55-1.58 (m, 1H), 1.42-1.44 (m, 1H),1.22-1.31 (m, 4H), 0.80 (t, J=6.8 Hz, 3H).

Example 11N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-morpholinothiazole-5-carboxamide

To a mixture of compound 3A (200 mg, 0.49 mmol) and DIPEA (171 μL, 0.98mmol) in THF (2 mL) was added morpholine (42 μL, 0.49 mmol). The mixturewas stirred for 1.5 h at 170° C. in a sealed Q-tube pressure reactor.Additional morpholine (42 μL, 0.49 mmol) was added and the mixture washeated for 0.5 h at 170° C. in a sealed Q-tube pressure reactor. Themixture was concentrated and the residue was purified by columnchromatography (0-5% MeOH/DCM) to give compound Example 11 (148 mg,73%). ¹H NMR (400 MHz, CDCl₃) δ 8.08 (br s, 1H), 7.64 (d, J=7.5 Hz, 1H),7.39 (s, 1H), 7.37 (d, J=8 Hz, 1H), 7.19 (dd, J=7.5 Hz, 1H), 7.12 (dd,J=7.5 Hz, 1H), 7.05 (d, J=2 Hz, 1H), 5.57 (d, J=8 Hz, 1H), 4.39-4.42 (m,1H), 3.80 (t, J=4.5 Hz, 4H), 3.51 (t, J=4.5 Hz, 4H), 3.06 (dd, J=14.5,5.5 Hz, 1H), 3.01 (dd, J=14.5, 5.5 Hz, 1H), 1.26-1.64 (m, 6H), 0.87 (t,J=7 Hz, 3H). ESMS+: 413.6 [M+1].

Examples 12-26 may be prepared according to the methods described above.

Example 27(S)—N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamideand Example 28(R)—N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide

Enantiomers were separated on a THAR 80 preparative SFC using aChiralpak AD-H column (250×30 mm; 5 μM id). The racemate was dissolvedin methanol (50 mg/mL) and 45 mg of racemate was loaded per injection.The separation was achieved using a mobile phase of 40% 2-propanol(additive: 0.05% NH₃H₂O) in CO₂ at a flow rate of 70 g/min and a systemback pressure of 100 bar. The column temperature was maintained at 40°C. and peaks were detected at 220 nm Total cycle time was 6 minutes.Example 27((S)—N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide):LCMS (Xtimate C18, 2.1×30 mm, 3 μM id): Peak 1 RT: 2.036 (100%) MS:426.2; Optical rotation (Dichrom Polaraizer, 589 nM) −0.143 (sd=0.0004);Chiral purity check (OJ-H, 40% MeOH (0.05% DEA)) Peak 1 RT: 3.61(99.87%), Peak 2 RT: 5.3 (0.13%). Example 28((R)—N-(1-(1H-Indol-3-yl)hexan-2-yl)-2-(4-methylpiperazin-1-yl)thiazole-5-carboxamide):LCMS (Xtimate C18, 2.1×30 mm, 3 μM id): Peak 1RT: 2.035 (98.77%) MS:426.2. Peak 2RT: 2.373 (1.23%) MS: 427.2; Optical rotation (DichromPolaraizer, 589 nM) +0.160 (sd=0.0003); Chiral purity check (OJ-H, 40%MeOH (0.05% DEA)) Peak 1 RT: 3.6 (0.18%), Peak 2 RT: 5.23 (99.82%).

Biological Example 1 In-Vitro Fluorescence Polarization Assay withAlpha-Synuclein Peptide Fragment (4F)

The fluorescence polarization assay tests the ability of compounds toinhibit the self-aggregation of α-synuclein peptide fragments. Peptideswere incubated for 60 min at room temperature in the presence or absenceof test compounds (compound concentrations were 33.3 to 0.3 μM). Sampleswere read on a BMG Pherastar plate reader in fluorescence polarizationmode using excitation at 485 nm and emission at 520 nm Data was analyzedusing a four-parameter logistic fit (XLFit, IDBS Software). Peptide 4F(CTGFVKKDQLGK (SEQ ID NO: 1)) was prepared by American Peptide. Freshpeptide samples were reconstituted in purified water at 5 mM and dilutedinto 50 mM HEPES pH 7.4 with 50 mM NaCl to 100 nM final concentration.Solid compounds were dissolved in DMSO (10 mM), and then diluted inbuffer.

Data for compounds tested are presented in Table 1. Comparativecompounds A and B were also tested.

TABLE 1 Ex. IC₅₀ (μM)* Comp. A >30^(§) Comp. B 6.35 1 3.33 ± 1.9^(¥) 23.55 3 0.27 4 5.9 5 1.3 6 0.39 7 0.37 8 0.77 9 8.5 27  0.4 ± 0.3^(§) 28 0.7 ± 0.4^(§) *n = 1 unless otherwise noted ^(§)n = 2 ^(¥)n = 3 ± SEM

Biological Example 2 In Vivo Pharmacokinetic Assays

The pharmacokinetics and brain distribution of the compounds describedherein was determined in male C57BL/6 mice following single intravenousor oral dose administration. A group of 54 male mice were divided intotwo groups of 27 mice. Animals in Group 1 (i.v.) and Group 2 (p.o.) weredosed with test compounds at 10 mg/kg (i.v.) or 2 mg/kg (p.o.). Bloodsamples were collected pre-dose and at 0.08, 0.25, 0.5, 1, 2, 4, 8, and24 h post-dose (i.v.), and pre-dose, and at 0.25, 0.5, 1, 2, 4, 6, 8 and24 h post-dose (p.o.). Blood was collected from sets of three mice ateach time point in labeled microcentrifuge tubes containing K₂EDTA asanticoagulant. Plasma samples were separated by centrifugation of wholeblood and stored below −70° C. until bioanalysis. After collecting bloodsamples, mice were humanely euthanized by CO₂ asphyxiation and brain wascollected at the same time points. Following collection, the brainsamples were washed in ice-cold phosphate buffer saline (pH 7.4), gentlydried on filter paper, weighed and placed in polypropylene tubes.Further brain samples were homogenized using phosphate buffer saline pH7.4 and the total homogenate volume was thrice the brain weight. Thesamples were then stored below −70° C. until bioanalysis. All sampleswere processed for analysis by protein precipitation using acetonitrileand analyzed with fit-for-purpose LC/MS/MS method (LLOQ: 1.01 ng/mL forplasma and brain). Pharmacokinetic parameters were calculated using thenon-compartmental analysis tool of Phoenix WinNonlin (Version 6.3).

Data obtained from this assay are presented in Table 2.

TABLE 2 Oral PK B:P Stability Plasma Bioavail- Clearance* ratio* Ex. inSGF Stability ablility* (mL/min/kg) (IV/PO) Comp. A stable  35% @ NC  71NC/0.05  60 min Comp. B unstable  89% @  14% 258   0.1/0.08  60 min    1stable 100% @  53%  81   2.0/0.4 120 min      4 stable  25% @ 100%  95  0.4/0.1 120 min     5 stable 100% @  79%  24  0.18/0.05 120 min   10ND ND  13%  75** 0.8**/0.15 27 ND ND  57%  37  1.4/ 0.3 28 ND ND  14% 69  2.1/ 0.4 * at 10 mg/kg ** at 2 mg/kg NC = No compound detected(below detection limit) ND = Not determined

Biological Example 3 NMR Assay for Effect of Test Compounds onAlpha-Synuclein Interaction with Lipid Membranes

To measure the interaction of test compounds with full-length ASYN inthe presence of lipid membranes, an NMR assay was conducted. NMRmeasurements were made in 20 mM Phosphate, pH=7.4, 100 mM NaCl on VarianDirect Drive 600 MHz and Varian Inova 800 MHz spectrometers with 10% D₂Oas lock solvent. Spectra were processed using NMRPipe (see F. Delaglio,S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, A. Bax, J Biomol NMR1995, 6, 277-293). α-Synuclein was used at 0.12 mM while1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)-liposomes wereadded at 0.8 mg/ml where present. All ¹H-¹⁵N correlation spectra wererecorded with a SOFAST pulse sequence (see P. Schanda, E. Kupce, B.Brutscher, J Biomol NMR 2005, 33, 199-211). Resonance assignment at nearphysiological conditions was readily available from a previouspublication (BMRB ID 16300; see J. N. Rao, Y. E. Kim, L. S. Park, T. S.Ulmer, J Mol Biol 2009, 390, 516-529). For ligand titration, Example 1was added stepwise to the liposome/ASYN mixture. ¹⁵N-¹H correlationspectra were recorded for each step and the signal intensities werereferenced to the free form of ASYN while accounting for dilutioneffects. To reduce noise in the available data, the intensity ratio forseveral amide positions of ASYN was averaged for two regions chosen tocorrespond to the SL1 and SL2 binding modes observed previously (see C.R. Bodner, A. S. Maltsev, C. M. Dobson, A. Bax, Biochemistry 2010, 49,862-871).

As shown in FIG. 1, the heteronuclear single quantum coherence (HSQC)spectroscopy signal intensity for ASYN is attenuated when ASYN isembedded in lipid membranes. This lipid-induced attenuation of the HSQCsignal was reversed by Example 1, thus demonstrating the ability of thetest compound to disrupt the association of ASYN with lipid membrane.FIG. 1A shows the signal attenuation as function of ASYN residues in thepresence of POPG liposomes. The Y axis (I/Io) is the ratio of the HSQCspectroscopy signal intensities for ASYN in the presence (I) or absence(Io) of lipid membranes. In FIG. 1B, the average I/Io ratio of ASYNresidues 3-23 was plotted as a function of the concentration of Example1 added. This plot shows that Example 1 reversed the interaction of ASYNwith 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (0.8mg/mL) liposomes in a concentration-dependent manner Similar resultswere obtained when ASYN residues 66-76 were analyzed.

Biological Example 4 Effect of Test Compounds on Annular Oligomers inLipid Membranes

Electron microscopy was used to directly visualize the effect of testcompounds on the formation of ASYN oligomers in lipid membranes. Formvargrids with the lipid monolayer were counterstained with a saturateduranyl acetate solution in 50% ETOH for 25 minutes. The grids were thenfloated on a droplet of 2% bismuth subnitrate for 10 min, and againcarefully rinsed with double distilled water three times and allowed tocompletely dry. Grids were imaged using a Zeiss EM10 transmissionelectron microscope Electron Microscope. From each sample grid, 5-10electron micrographs at 10,000× magnification and 5-10 images at 40,000×were obtained. The best negatives were scanned and analyzed with theImageJ 1.43 program to estimate the numbers of annular oligomers perhigher power field (100×100 nm) (Rasband, W. S., ImageJ, U. S. NationalInstitutes of Health, Bethesda, Md., USA, http://imagej.nih.gov/ij/,1997-2014).

In this study, Example 1 was found to dramatically reduce theaccumulation of annular, ring-like, oligomeric forms of ASYN in a lipidmembrane, while small non-annular aggregates were still observed. FIG.2A shows electron microscopic images of ASYN oligomers formed on lipidcoated Formvar grids in the absence and presence of Example 1. FIG. 2Bis a graph that reflects the quantification of the electron microscopicimages. Example 1 reduced the number of annular ASYN oligomers detectedon the Formvar grids at concentrations as low as 10 nM (means±SEM for 20measurements). Example 1 achieved these effects at sub-stoichiometricconcentrations relative to ASYN. These findings were consistent with themolecular dynamic modeling showing that Example 1 stabilizesconformations of ASYN less likely to form ring-like oligomers in lipidmembranes.

These results suggest that Example 1 interacts with oligomeric andlipid-bound forms of ASYN in a way that reduces the affinity of ASYNoligomers for the lipid membrane. Example 1 was able to interfere withASYN oligomerization, the binding of ASYN to lipid membranes, and theformation of annular ring-like oligomers (“pores”) in these membranes.These results also suggest that Example 1 alters the aggregation of ASYNand prevents the formation of specific oligomeric structures believed tocontribute to the neurotoxicity of misfolded, oligomerized ASYN inParkinson's disease.

Biological Example 5 Effect of Test Compounds on Alpha-Synuclein inCells

The effect of Example 1 on accumulation of ASYN in B103 neuroblastomacells overexpressing human ASYN was studied. A lentiviral expressionsystem was used to express GFP-tagged ASYN in these cells. Forty-eighthours after expression was initiated, vehicle or Example 1 (0.1 or 1.0μM) was added for an additional 24 hours. The amount of accumulatedGFP-ASYN was then visualized. As shown in FIG. 3, Example 1 reduced theintensity of GFP florescence in these cells at 1.0 μM (*p<0.05 vs.vehicle control group). Example 1 was therefore found to reduceconcentrations of ASYN-GFP in ASYN-overexpressing cells.

Biological Example 6 In Vivo Efficacy Studies

Parkinson's disease (PD) is characterized by aberrant accumulation ofoligomeric forms of alpha-synuclein (ASYN). It is hypothesized thatthese toxic forms of ASYN contribute to the neuronal dysfunction andcell death observed in PD and other synucleinopathies, in part, thoughthe formation of pore-like structures in cell membranes. The compoundsdescribed herein were designed to ameliorate PD-related symptoms andpathology by selectively blocking the formation and accumulation ofthese toxic species of ASYN.

A) Transgenic Mouse Model of Parkinson's Disease

Example 1 was evaluated in a transgenic mouse model of PD overexpressinghuman wild-type ASYN under the Thy-1 promoter (also referred to as theLine 61 ASYN transgenic mouse), by administering Example 1 aTant 0, 1,or 5 mg/kg (i.p.) once daily (five days per week) for three months andthen assessing PD-relevant sensorimotor performance, biochemicalalterations, and neuropathological changes in ASYN and related proteins.

The Round Beam Task was used to assess sensorimotor impairments, usingnumber of slips as the primary outcome measure (FIG. 4). To confirm theviability of the transgenic mouse model, ASYN transgenic andnon-transgenic mice were tested, and the number of slips forvehicle-treated transgenic subjects was statistically significantlyhigher than in the vehicle-treated non-transgenic control group(****p<0.0001). Transgenic mice treated with Example 1 at both testdoses had statistically significantly reductions in slips compared tovehicle-treated transgenic mice (^(#)p<0.05 and ^(##)p<0.01 vs.vehicle-treated ASYN transgenic mice).

Western Blot analysis of cerebral cortical and hippocampal brainhomogenates revealed statistically significant reductions in transgenicASYN protein levels. Biochemical evaluations of oligomeric proteinsusing A11 antibody dot blot methods (including ASYN) in corticalhomogenates were performed. The transgenic mouse model was verified, asA11 antibody dot blot evaluation of oligomers in cortical homogenatesshowed a statistically significant increase in A11 immunostaining in thecytosolic fraction of the frontal cortex in vehicle-treated ASYNtransgenic mice relative to vehicle-treated non-transgenic control mice(FIG. 5; *p<0.05). Treatment with Example 1 (5 mg/kg) yielded astatistically significant decrease in oligomers in the cytosolicfraction from the frontal cortex region of mice relative tovehicle-treated ASYN transgenic mice (FIG. 5; ^(###)p<0.001).

B) Line 61 ASYN Transgenic Mouse Models

Previous immunolabeling studies by Masliah and colleagues havedemonstrated statistically significant increases in ASYN immunolabelingin cortical neuropil in the Line 61 ASYN transgenic mouse (Masliah E. etal., Science, 2000, 287(5456):1265-9). These neuropathological findingswere reconfirmed in the current study using the methods described byMasliah and colleagues. Example 1 administration (1 and 5 mg/kg dosing)produced statistically significant decreases in ASYN levels asdetermined by effects on ASYN immunolabeling (FIGS. 6 and 7). There wasa statistically significant increase in ASYN immunolabeling with theMillipore anti-alpha-synuclein antibody in the cortical neuropil(****p<0.0001) (FIG. 6A) and in neuronal cell bodies of ASYN transgenicmice (**p<0.01) (FIG. 6B) relative to non-transgenic/vehicle controls.Example 1 (1 and 5 mg/kg) administration produced statisticallysignificant decreases in alpha-synuclein immunolabeling in corticalneuropil (FIG. 6A) (^(####)p<0.0001 vs. vehicle-treated ASYN transgenicmice), and a non-statistically significant decrease in the number ofASYN immunolabeled neuron cell bodies at 5 mg/kg (FIG. 6B).

Moreover, normalization of neurodegeneration-related markers includingtyrosine hydroxylase, NeuN, and GFAP were observed.

As shown in FIG. 8, the effect of Example 1 at 0.5 mg/kg and 1 mg/kg onsensorimotor impairment in Line 61 ASYN transgenic mice was studied,using the Round Beam Motor Performance assay described above. Astatistically significant increase in the number of slips was observedin vehicle-treated ASYN transgenic control mice as compared tovehicle-treated non-transgenic control subjects (****p<0.0001). At 1mg/kg, Example 1 treatment produced a statistically significantimprovement (decreased slips) in ASYN transgenic mice relative tovehicle-treated ASYN transgenic mice (^(##)p<0.01). At 0.5 mg/kg,Example 1 produced a non-statistically significant decrease in slips.

Together, these results demonstrate that Example 1 significantlyimproves sensorimotor, biochemical, and neuropathological outcomes in atransgenic mouse model. These findings confirmed that administration ofExample 1 produces improvements in behavioral, biochemical, andneuropathological measures in a transgenic mouse model of Parkinson'sdisease/Dementia with Lewy bodies (PD/DLB).

Biological Example 7 Development of Biological Markers

A) Fecal Boli Counts

In efforts directed toward development of translatable functional andbiochemical biomarkers, additional evaluations were conducted, includingan assessment of fecal boli counts produced in a novel environment andpost-mortem cardiac levels of ASYN.

Chronic constipation in Parkinson's patients has a prevalence of 50-80%,and may precede diagnosis by 20+ years (Awad, R. A. World J.Gastroenterol. 2011, 17(46), 5035-5048; Kim, J. S. et al., J. Neurol.Sci. 2011, 310(1-2), 144-151). Associated symptoms include decreasedtransit times and EMG abnormalities (sphincter, rectoanal inhibitoryreflexes), and are accompanied by key pathological findings includingLewy bodies in parasympathetic nuclei and nerves, and decreaseddopaminergic neurons. This bowel dysfunction is compounded by decreasedactivity levels, changes in diet (food and water), and effects ofParkinson's medications.

An earlier published study included a report that Line 61 ASYNtransgenic mice have decreased colonic motility, fecal output, andweight (Wang, L. et al. Neurogastroenterol. Motil. 2012, 24(9),e425-436). For the present study, an assessment of fecal boli counts wasconducted in conjunction with a spontaneous locomotor activity testsession. At the conclusion of a five-minute test session, theexperimenter counted the number of fecal boli present in the testchamber, and the results are presented in FIG. 9. Vehicle-treated ASYNtransgenic mice had statistically significant reductions in fecal boliproduced in a novel environment relative to vehicle-treatednon-transgenic control mice (*p<0.05). Example 1 had no effect on numberof boli produced in non-transgenic mice, but restored function in ASYNtransgenic mice at 0.5 mg/kg (#p<0.05 vs. vehicle-treated ASYNtransgenic mice) and 1 mg/kg (^(###)p<0.001 vs. vehicle-treated ASYNtransgenic mice).

B) Cardiac Function

Like bowel dysfunction, alterations in cardiac biochemistry and functionmay precede Parkinson's disease diagnosis by 20 years or more.Well-characterized functional alterations in PD patients include alteredheart rate variability and orthostatic hypotension (Kaufmann, H. et al.,Handbook Clin. Neurol. 2013, 117, 259-278; Jain, S. et al., Neurobiol.Dis. 2012, 46(3), 572-580; Senard, J. M. et al., Rev. Neurol. (Paris)2010, 166(10), 779-784; Post, K. K. et al., Parkinsonism Relat. Disord.2008, 14(7), 524-531). These functional changes are accompanied bypathological findings of loss of myocardial noradrenergic innervationand the presence of ASYN aggregates in cardiac autonomic nerves(Jellinger, K. A., J. Neurol. Sci. 2011, 310(1-2), 107-111). Previouscharacterizations of cardiac function and biochemistry in Line 61 ASYNtransgenic mice have demonstrated the presence of hASYN in theventricular and atrial walls of the heart localized within noradrenergicfibers (Fleming, S. M., J. Parkinsons Dis. 2011, 1(4), 321-327). For thepresent study, post-mortem Western blot evaluations of cardiac ASYN byWestern blot analysis were performed to confirm the presence in ASYNtransgenic cardiac tissue and to evaluate the effects of Example 1 ontransgenic cardiac levels of ASYN (FIG. 10). There was a statisticallysignificant increase in detected cardiac levels of ASYN invehicle-treated ASYN transgenic mice relative to vehicle-treatednon-transgenic control mice (***p<0.001). There were statisticallysignificant normalizations of ASYN levels in ASYN transgenic micetreated with either 0.5 or 1 mg/kg of Example 1 relative tovehicle-treated ASYN transgenic mice (####p<0.0001).

C) Retinal Imaging

Abnormal accumulation of a neuronal protein alpha-synuclein (ASYN) hasbeen hypothesized to underlie neuronal cell death and synapticdysfunctional in Parkinson's disease (PD) and Dementia with Lewy Bodies(DLB). Compounds that selectively interfere with alpha-synucleinprotein-folding dynamics and prevent the formation of propagating dimershave been developed and further evaluated in animal models. Alterationsin visual function are present in some Parkinson's patients (Botha, H.et al., Parkinsonism Relat. Disord. 2012, 18(6), 742-747; Bodis-Wollner,I. et al., Behav. Neurosci. 2013, 127(2), 139-150; Bodis-Wollner, I.,Parkinsonism Relat. Disord. 2013, 19(1), 1-14; Javaid, M. A. et al.,Parkinsonism Relat. Disord. 2012, 18(Suppl. 1), S100-3) and recentreports have presented potential pathological changes in PD retinae.Optical coherence tomography (OCT) studies have demonstrated a decreasedretinal nerve fiber layer in Parkinson's patients (Yu, J. G. et al.,PLoS One 2014, 9(1), e85718). Post-mortem assessments have revealed ASYNdeposits in PD retina (Bodis-Wollner, I. et al., Ann. Neurol. 2014,75(6), 964-6).

The feasibility of repeated longitudinal retinal imaging evaluations ofe-GFP-ASYN in the PDNG78 transgenic mouse model of DLB/PD wasdemonstrated previously as a method to evaluate and track theprogression of neurodegenerative changes in animal models of Parkinson'sdisease (Rockenstein et al., “Retinal scanning evaluations ofalpha-synuclein-eGFP deposition in a transgenic mouse model of PD/DLB,”Society for Neurosciences, Annual Meeting, 2013, Abstract No. 329.06).Progressive pathological features in the PDNG78 retina were shown tomirror CNS pathology, thereby providing a means to non-invasively andrepeatedly evaluate potential therapeutic interventions in a transgenicmouse model of PD/DLB.

This study was conducted to determine the effect of Example 1 (3 monthsi.p. administration at 0 & 5 mg/kg) on the presence and progression ofalpha-synuclein (ASYN) retinal pathology in a longitudinal retinalimaging study in a transgenic mouse model of Parkinson'sdisease/Dementia with Lewy Bodies (PD/DLB). The transgenic mice subjectsoverexpress fused alpha-synuclein-GFP (green fluorescent protein) underthe PDGF-beta promoter and are commonly referred to as PDNG78 transgenicmice (Rockenstein, E. et al., J. Neurosci. Res. 2005, 80, 247-259). ThePDNG78 transgenic mouse expresses fused ASYN-GFP at levels 2-5 foldgreater levels than non-transgenic control mice. The CNS expressionlevels of ASYN are highest in the limbic system, including the neocortexand hippocampal regions, of PDNG78 transgenic mice. Cellulardistributions of ASYN-GFP mirror synucleinopathy-relevant featuresincluding accumulations in neuronal cell bodies, diffuse staining of theneuropil, synaptic punctate staining, and perivascular deposits.

A total of four imaging sessions were conducted, including a baselineprior to starting treatments and three subsequent imaging sessions atapproximately one-month intervals. Analysis of retinal images forpercentage of image with ASYN-GFP (FIG. 11) showed statisticallysignificant increases in the percentage of image areas with ASYN-GFP inthe retinae of transgenic mice at baseline prior to commencement oftreatments (**p<0.01 vs. vehicle-treated non-transgenic mice) and foreach subsequent scan (*p<0.05 and ***p<0.001 vs. vehicle-treatednon-transgenic mice). The percentage of ASYN-GFP positive area decreasedin transgenic mice treated with Example 1 (5 mg/kg) after approximately60 days of treatment and persisting through the 90 day imaging timepoint (###p<0.001 vs. vehicle-treated ASYN transgenic mice).

Analysis of ASYN-GFP positive particle counts (FIG. 12) revealedincreased and persistent perivascular and nerve terminal greenfluorescent protein (GFP) labeling in transgenic, but not non-transgenicmice. There were statistically significant increases in total ASYN-GFPparticle counts in the retinae of transgenic mice at baseline prior tocommencement of treatments (*p<0.05 vs. vehicle-treated non-transgenicmice) and for scans starting at approximately 60 days of treatments(**p<0.01 vs. vehicle-treated non-transgenic mice). The number ofASYN-GFP positive particles was decreased in ASYN transgenic micetreated with Example 1 (5 mg/kg) after approximately 60 days oftreatment and persisting through the 90 day imaging time point (##p<0.01vs. vehicle-treated ASYN transgenic mice).

Findings from this study demonstrate that administration of Example 1 (5mg/kg per day; for 3 months) produces beneficial changes in ASYN retinalpathology of transgenic mice overexpressing ASYN as a model of PD/DLB.These data also provide additional evidence measure of beneficialeffects of Example 1 by a potentially translatable imaging method.

The invention claimed is:
 1. A compound that is:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising (a) the compound or pharmaceutically acceptablesalt thereof according to claim 1, and (b) a pharmaceutically acceptableexcipient.
 3. The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 4. The compound of claim1, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising (a) the compound or pharmaceutically acceptablesalt thereof according to claim 3, and (b) a pharmaceutically acceptableexcipient.
 6. A pharmaceutical composition comprising (a) the compoundor pharmaceutically acceptable salt thereof according to claim 4, and(b) a pharmaceutically acceptable excipient.